Cell Signaling: Communication Between Cells And Their Environment

Objectives

• Outline the general characteristics of signal transduction systems.

• Describe the mechanism by which G protein coupled-receptors produce secondary messengers.

• Describe the experiments leading up to the discovery of second messengers and the role of cAMP.

• Outline the chain of events in the protein kinase initiated reaction cascade leading to glucose release.

• Describe the points at which cascades can be regulated by phosphatases and inhibitors.

• Outline the function and structure of G proteins and the receptors with which they interact.

• Describe phosphatidylinositol-derived second messengers and the functions of Ca2+ as a second messenger and the downstream effectors that both activate.

• Elaborate upon the characteristics of the receptor tyrosine kinase signaling pathway.

• Outline insulin's mechanism of action; stress the involvement of RTKs and SH2-containing proteins.

• Describe the roles of the Ras protein and the MAP kinase cascade in the control of cell function.

• Illustrate the differences between the convergence, divergence and crosstalk among different signaling pathways.

• Outline the roles of carbon monoxide and nitric oxide as cellular messengers.

• Describe the way in which integrins and other membrane receptors interact with the extracellular matrix and subsequently convey messages to the cytosol.

• Acquaint students with the concept of apoptosis and present them with examples to illustrate it.

• Compare and contrast the signaling pathways in plants with those in animals.

Essential Concepts Cell Signaling

1. All cells are equipped with elaborate systems for receiving signals from their environment. The 

2. mechanisms involved in cell signaling are particularly crucial for understanding multi-cellular organisms, and are an important in many diseases. Signaling systems are providing new targets for treating disease.Cells receive signals from their environment (light, odor, sound, nutrients) and other cells (divide, differentiate, migrate, transmit signals).

3. Signaling involves a receptor, the transduction of information from the cell surface to inside the cell, and subcellular systems that make a response.

4. Receptors are proteins with specific binding sites for signaling molecules called ligands. Membrane soluble ligands include steroid hormones (estrogen) which bind to cytoplasmic receptors. Most other signaling molecules (peptides, proteins, gases, ions, nucleotides) bind to integral membrane proteins.

5. Types of receptors include ion channels that can open or close (gated) allowing ions to enter or leave cells, enzyme linked that activate endogenous or exogenous protein kinases (phosphate transferring enzymes), and serpentine or G-protein-linked.

6. Ligand binding to receptor results in the production of second messengers (cAMP, cGMP, IP3, Ca++, DAG, NO). Activation frequently involves protein kinase cascades, a series of amplification steps resulting in a rapid response.

7. Cells can have similar receptors, but make very different responses to the same ligand. The response made depends on the availability of targets. One cell may have target proteins that leads to altered transcription, while another has target proteins that leads to cell differentiation or division.

8. Animal and plant cells have membrane proteins that permit direct cell to cell communication by opening channels between cell cytoplasms. These are called gap junctions in animal cells and plasmodesmata in plant cells.

Lecture Outline

Cell Signaling: Introduction

I. To survive, cells must communicate with their neighbors, monitor environmental conditions & respond appropriately to a host of different stimuli that impinge on their surface

A. Most cells in a complex multicellular organism are specialized to carry out ≥1 specific functions

B. Many biological processes require various cells to work together & to coordinate their activities – to do this, cells must communicate, which is accomplished by a process called cell signaling

II. Cell signaling makes it possible for cells to talk to each other & for an organism to function as a coherent system; it affects virtually every aspect of cell structure & function

A. An understanding of cell signaling requires knowledge about other types of cellular activity

B. Insights into cell signaling can tie together a variety of seemingly independent cellular activities

C. Cell signaling is also intimately involved in regulation of cell growth & division, making it important in understanding the development of a malignant tumor

The Basic Elements of Cell Signaling Systems in the Body

I. Cells usually communicate with each other through extracellular messenger molecules; there are 3 types of signaling & signaling molecules

A. Autocrine signaling – cell producing the messenger expresses receptors on its surface that can respond to that messenger; thus, cells releasing the message will stimulate (or inhibit) themselves

B. Paracrine stimulation – messenger molecules travel only short distances through the extracellular space to cells that are in close proximity to the cell that is generating the message

    1. These messenger molecules are usually limited in their ability to travel around the body because they are inherently unstable or they are degraded by enzymes or they bind to extracellular matrix

C. Endocrine signaling – messenger molecules reach their target cells via passage through bloodstream; these messengers are also called hormones & typically act on target cells located at distant sites in body

II. Cell signaling is initiated with the release of a messenger molecule by a cell engaged in sending messages to other cells in body

A. Extracellular messengers can travel a short distance & stimulate cells that are in close proximity to the origin of the message

    1. Sometimes the messenger molecule need only diffuse across a narrow cleft or through a tiny blood vessel before the message is received by an appropriate target cell

B. Other times, the messenger molecule may have to circulate through the entire body before reaching & stimulating specific target cells that are far away from source

III. Cells can only respond to an extracellular message if they express receptors that specifically recognize & bind that particular messenger molecule

A. Usually, the messenger molecule (or ligand) binds to a receptor at the extracellular surface of the responding cells

B. Binding of the ligand to the extracellular surface of the receptor relays a signal across the membrane to the receptor's cytoplasmic domain at the inner membrane surface

IV. Once it has reached the inner plasma membrane surface, there are 2 major routes by which the signal is transmitted into the cell interior; the particular route depends on the type of receptor activated

A. One type of receptor transmits signal from its cytoplasmic domain to a nearby enzyme that generates a second messenger

    1. Since it brings about (effects) the cellular response by generating a second messenger, the enzyme responsible is called an effector

    2. Second messengers are small substances that typically activate (or inactivate) specific proteins

    3. Depending on its chemical structure, a second messenger may diffuse through the cytosol or remain embedded in the membrane lipid bilayer

B. Another type of receptor transmits a signal by transforming its cytoplasmic domain into a recruiting station for cellular signaling proteins

V. Whether the signal is transmitted by a second messenger or by protein recruitment, the outcome is similar, a protein that is positioned at the top of an intracellular signaling pathway is activated

A. Each signaling pathway consists of a series of distinct proteins that operate in sequence

B. Each protein in the pathway typically acts by altering the conformation of the subsequent (downstream) protein in the series, an event that activates or inhibits the protein

C. Alterations in the conformations of signaling proteins are often accomplished by protein kinases & protein phosphatases that, respectively, add or remove phosphate groups from other proteins

    1. Human genome encodes >500 different protein kinases & >100 different protein phosphatases; some kinases & phosphatases are soluble cytoplasmic proteins, others integral membrane proteins

    2. Some have numerous proteins as their substrates; others phosphorylate or dephosphorylate only a single amino acid residue of a single protein substrate

D. Many of the protein substrates of these enzymes are enzymes themselves, like other kinases & phosphatases, but include ion channels, transcription factors & various types of regulatory proteins

E. It is thought that at least 50% of transmembrane & cytoplasmic proteinsare phosphorylated at one or more sites; protein phosphorylation can change protein behavior in several different ways:

    1. It can activate or inactivate an enzyme

    2. It can increase or decrease protein-protein interactions

    3. It can induce a protein to move from one subcellular compartment to another or

    4. It can act as a signal that initiates protein degradation

F. Many of the protein kinases & their target proteins have been identified; the primary challenge is to understand the roles of these diverse posttranslational modifications in activities of different cell types

G. Signals transmitted along such signaling pathways ultimately reach target proteins involved in basic cell processes; depending on cell type & message, response initiated by target protein may involve:

    1. A change in gene expression

    2. An alteration of the activity of metabolic enzymes

    3. A reconfiguration of the cytoskeleton

    4. An increase or decrease in cell mobility

    5. A change in ion permeability

    6. Activation of DNA synthesis or

    7. Even the death of the cell

H. Virtually every activity in which a cell is engaged is regulated by signals originating at cell surface

    1. The overall process in which information carried by extracellular messenger molecules is translated into changes that occur inside of a cell is called signal transduction

VI. Finally, signaling has to be terminated so that cells can be responsive to additional messages that they may receive

A. First, they must eliminate the extracellular messenger molecule – certain cells produce extracellular enzymes that destroy specific extracellular messengers

B. In other cases, activated receptors are internalized; once internalized, the receptor may be degraded together with its ligand, leaving the cell with decreased sensitivity to subsequent stimuli

C. Alternatively, receptor & ligand may be separated within an endosome, after which the ligand is degraded & the receptor is returned to the cell surface

A Survey of Extracellular Messengers and Their Receptors

    I. A large variety of molecules can function as extracellular carriers of information, including:

A. Amino acids and amino acid derivatives – glutamate, glycine, acetylcholine, epinephrine, dopamine & thyroid hormone; these molecules act as neurotransmitters & hormones

B. Gases, such as NO & CO

C. Steroids, which are derived from cholesterol – steroid hormones regulate sexual differentiation, pregnancy, carbohydrate metabolism, & excretion of sodium & potassium ions

D. Eicosanoids – nonpolar molecules containing 20 carbons that are derived from a fatty acid named arachidonic acid

    1. They regulate a variety of processes including pain, inflammation, blood pressure & blood clotting

    2. Several over-the-counter drugs used to treat headaches & inflammation inhibit eicosanoid synthesis

E. A wide variety of polypeptides & proteins

    1. Some are present as transmembrane proteins on the surface of an interacting cell

    2. Others are part of, or associate, with the extracellular matrix

    3. Finally, many proteins are excreted in the extracellular environment where they are involved in regulating processes like cell division, differentiation, immune response or cell death & cell survival

II. Extracellular signaling molecules are usually, but not always, recognized by specific receptors that are present on the surface of the responding cell

A. Receptors bind their signaling molecules with high affinity

B. They then translate this interaction at cell's outer surface into changes that take place on inside of cell

III. The receptors that have evolved to mediate signal transduction are:

A. G-protein coupled receptors (GPCRs) – huge family of receptors that contain 7 transmembrane -helices

    1. They translate binding of extracellular signaling molecules into activation of GTP-binding proteins

    2. GTP-binding proteins (G proteins) are involved in vesicle budding & fusion, MT dynamics, protein synthesis & nucleocytoplasmic transport & transmitting messages along cell information circuits

B. Receptor protein-tyrosine kinases (RTKs) – a second class of receptors that have evolved to translate the presence of extracellular messenger molecules into changes inside the cell

    1. Binding of specific extracellular ligand to RTK usually results in receptor dimerization followed by activation of receptor's protein-kinase domain, which is present within its cytoplasmic region

    2. Upon activation, these protein kinases phosphorylate cytoplasmic substrate proteins, thereby altering their activity, their localization or their ability to interact with other proteins within the cell

    3. Most protein kinases transfer phosphate groups to serine or threonine residues of their protein substrates, but RTKs phosphorylate tyrosine residues

C. Ligand-gated channels – cell surface receptors that bind to extracellular ligands; their ability to conduct a flow of ions across the plasma membrane is regulated directly by ligand binding

    1. Flow of ions across membrane can result in temporary change in membrane potential

    2. This will affect the activity of other membrane proteins, like voltage-gated channels

    3. This is the basis for the formation of the nerve impulse; also the influx of Ca2+ ions can change the activity of particular cytoplasmic enzymes; they function as receptors for neurotransmitters

D. Steroid hormone receptors function as ligand-regulated transcription factors

    1. Steroid hormones diffuse across plasma membrane & bind to their receptors, which are present in the cytoplasm

    2. Hormone binding results in conformational change that causes hormone-receptor complex to move into nucleus & bind to elements present in promoters or enhancers of hormone responsive genes

    3. This interaction gives rise to an increase or decrease in the rate of gene transcription

E. Several types of receptors act by unique mechanisms

    1. Some, like the B- & T-cell receptors that are involved in the response to foreign antigens, associate with known signaling molecules such as cytoplasmic protein-tyrosine kinases

    2. For others, the mechanism for signal transduction remains to be established

G Protein-Coupled Receptors: Background Information on Receptors

I. G-protein coupled receptors (GPCRs) – are so-named because they interact with G proteins; also referred to as seven-transmembrane (7TM) receptors because they contain 7 transmembrane helices

A. Thousands of different GPCRs have been identified in organisms ranging from yeast to flowering plants & mammals; they regulate an extraordinary spectrum of cellular processes

    1. They constitute the single largest protein superfamily encoded by animal genomes

B. Included among the natural ligands that bind to GPCRs are a diverse array of hormones, chemoattractants, neurotransmitters, opium derivatives, odorants, tastants & photons

    1. Chemoattractants, an example of which are molecules that attract phagocytic cells of immune system

    2. Odorants & tastants are molecules detected by the olfactory & gustatory receptors eliciting the senses of smell & taste

II. G protein-coupled receptors normally have the following topology:

A. Their amino-terminus is present on the outside of the cell

B. The 7 -helices that traverse the plasma membrane are connected by loops of varying length

C. Carboxy-terminus is present on the inside of the cell

    1. There is a growing number of proteins that bind to GPCR carboxy-termini

    2. Many of these proteins act as molecular scaffolds that link receptors to various signaling proteins & effectors present in the cell

D. There are 3 loops present on the outside of the cell that, together, form the ligand-binding site

E. There are also 3 loops present on the cytoplasmic side of the plasma membrane that provide binding sites for intracellular signaling proteins

    1. G proteins bind to the third intracellular loop

    2. Arrestins also bind to the third intracellular loop & compete with G proteins for binding to receptor

III. The allosteric model holds that GPCRs can exist in an active & inactive conformation

A. The inactive conformation is stabilized by noncovalent interactions between transmembrane helices

    1. Ligand binding disturbs these interactions thereby causing the receptor to assume an active conformation

    2. This requires rotations & shifts of the transmembrane helices relative to each other

    3. Because they are attached to the cytoplasmic loops, rotation or movement of these transmembrane helices relative to each other causes changes in the conformation of the cytoplasmic loops

B. This, in turn, leads to an increase in the receptor's affinity for a G protein that is present on the cytoplasmic surface —> the ligand-bound receptor forms a receptor-G protein complex

C. The interaction with the receptor induces a conformational shift in the subunit of the G protein, causing the release of GDP, which is followed by the binding of GTP —> G protein is activated

    1. While in the activated state, a single receptor can activate a number of G protein molecules, providing a means of signal amplification

G Protein-Coupled Receptors: G Proteins – General Structure and Function

I. Heterotrimeric G proteins - discovered, purified, & characterized by Martin Rodbell et al (NIH) & Alfred Gilman et al (Univ. of Va.) – called G proteins since they bind guanine nucleotides, either GDP or GTP

A. They are described as heterotrimeric because all of them consist of 3 different polypeptide subunits (, and ), distinguishing them from small, monomeric G proteins, like Ras

B. Heterotrimeric G proteins are held at the plasma membrane by lipid chains that are covalently attached to the & subunits

C. The guanine-nucleotide-binding site is present on the G subunit

II. Replacement of GDP by GTP after an interaction with an activated GPCR causes a conformational change in the G subunit

A. In its GTP-bound conformation, the Gsubunit has a low affinity for G, leading to its dissociation from the complex

B. Each dissociated Gsubunit with GTP attached is free to activate an effector protein like adenylyl cyclase, which in this case leads to the production of the second messenger, cAMP

1. Other effectors include phospholipase C- and cyclic GMP phosphodiesterase

C. Second messengers, in turn, activate one or more cellular signaling proteins

III. A G protein is said to be "on" when it’s a subunit is bound to GTP; G subunits can turn themselves off by hydrolysis of GTP to GDP & inorganic phosphate (Pi)

A. This results in a conformational change causing a decrease in the affinity for the effector & an increase in the affinity for the subunit

1. Thus, after GTP hydrolysis, the G subunit will dissociate from the effector & reassociate with the subunit to reform the inactive heterotrimeric G protein

B. In a sense, heterotrimeric G proteins function as molecular timers

1. They are turned on by the interaction with an activated receptor & turn themselves off by hydrolysis of bound GTP after a certain amount of time has passed

2. While they are active, Gsubunits can turn on downstream effectors

IV. Heterotrimeric G proteins come in 4 flavors: Gs, Gq, GI, & G12/13 – this classification is based on the G& the effectors to which they couple

A. Gs family members couple receptors to adenylyl cyclase (activated by GTP-bound G subunits)

B. Gq family members contain Gsubunits that activate phospholipase C- (PLC) - PLC hydrolyzes phosphatidylinositol diphosphate, producing inositol triphosphate & diacylglycerol

C. Activated GI subunits function by inhibiting adenylyl cyclase

D. G12/13 members are less well characterized than the other G protein families, although their inappropriate activation has been associated with excessive cell proliferation & malignant transformations

V. After its dissociation from the G subunit, the complex also has a signaling function & it can couple to at least 4 different types of effectors: PLC, K+ ion channels, adenylyl cyclase & PI 3-kinase

G Protein-Coupled Receptors: G Proteins - Response Termination

I. To prevent overstimulation, receptors must be blocked from continuing to activate G proteins; to regain sensitivity to future stimuli, receptor, G protein & effector must all return to inactive state

II. Desensitization – the process that blocks active receptors from turning on additional G proteins; it takes place in 2 steps

A. Step 1 of desensitization – the cytoplasmic domain of the activated receptor GPCR is phosphorylated by a specific type of kinase, G protein-coupled receptor kinase (GRK)

1. GRKs form a small family of serine-threonine protein kinases

2. The conformational changes that make it possible for GPCRs to activate G proteins also make them good GRK substrates; as a result, GRKs specifically recognize activated GPCRs

B. Step 2 – GPCR phosphorylation sets the stage for second step, which is the binding of proteins (arrestins)

1. Arrestins form a small group of proteins that bind GPCRs & compete for binding with heterotrimeric G proteins —> thus, arrestin binding prevents further activation of additional G proteins

2. This is termed desensitization because the cell stops responding to the stimulus, while that stimulus is still acting on the outer surface of the cell

3. Desensitization is one of the mechanisms that allows a cell to respond to a change in its environment, rather than continuing to fire endlessly in the presence of an unchanging environment

4. Importance of desensitization - mutations interfering with rhodopsin phosphorylation by a GRK lead to retinal photoreceptor cell death (thought to be one cause of blindness due to retinitis pigmentosa)

III. While bound to phosphorylated GPCRs, arrestin molecules are also capable of binding to clathrin molecules that are situated in clathrin-coated pits

A. The interaction between bound arrestin & clathrin promotes the uptake of phosphorylated GPCRs into the cell by endocytosis

1. Depending upon circumstances, receptors that have been removed from the surface by endocytosis may be dephosphorylated & returned to the plasma membrane

2. Alternatively, internalized receptors are degraded in lysosomes

B. If the receptors are degraded, the cells lose, at least temporarily, sensitivity for the ligand in question; if receptors are returned to the cell surface, the cells remain sensitive to the ligand

IV. Signaling by the activated Gsubunit is terminated by a less complex mechanism: the bound GTP molecule is simply hydrolyzed to GDP

A. Thus, strength & duration of the signal are determined partly by the G subunit GTP hydrolysis rate

1. G subunits have weak GTPase activity, allowing them to slowly hydrolyze bound GTP, inactivating themselves

B. Termination of the response is accelerated by regulators of G protein signaling (RGSs)

1. The interaction with an RGS protein increases the rate of GTPase hydrolysis by the G subunit

2. Once the GTP is hydrolyzed, the G-GDP reassociates with the G subunits to reform the inactive trimeric complex —> returns system to the resting state

G Protein-Coupled Receptors: G Proteins – Signal Transmission Across Membrane

I. Mechanism for transmitting signals across the plasma membrane by G proteins is of ancient evolutionary origin & is highly conserved

A. Yeast cells were genetically engineered to express a receptor for the mammalian hormone somatostatin

B. When these yeast cells were treated with somatostatin, the mammalian receptors at the cell surface interacted with yeast heterotrimeric G proteins at the inner membrane surface

1. Response leads to triggering of yeast cell proliferation

II. Bacterial toxins – because G proteins are so important to the normal physiology of multicellular organisms, they have been targeted by bacterial pathogens

A. Cholera toxin (produced by Vibrio cholerae) exerts its effect by modifying G-subunits & inhibiting their GTPase activity in the cells of the intestinal epithelium; thus, the G

1. The G bind to adenylyl cyclase molecules, which remain in activated mode, churning out cAMP, which causes epithelial cells to secrete large volumes of fluid into the intestinal lumen

2. The loss of water associated with this inappropriate response often leads to death due to dehydration

B. Pertussis toxin is one of several virulence factors produced by Bordetella pertussis, a microorganism that causes whooping cough

1. Whooping cough is a debilitating respiratory tract infection seen in 50 million people worldwide each year, causing death in ~350,000 of these cases annually

2. Pertussis toxin also acts by inactivating G-subunits, which interferes with the signaling pathway that leads the host to mount a defensive response against the bacterial infection

G Protein-Coupled Receptors: The Discovery of Second Messengers – Cyclic AMP, A Prototypical Second Messenger

I. The discovery of 2nd messenger: cyclic AMP – mid-1950s; Earl Sutherland et al. (Case Western Reserve); Edwin Krebs & Edmond Fischer (U. of Washington); how a hormone alters cytoplasmic enzyme activity

A. Try to determine physiological responses to hormone; studied with in vitro system of broken cells

1. Eventually, they activated phosphorylase in a broken-cell prep exposed to glucagon or epinephrine

2. Divided broken-cell preparation into particulate fractions (mostly cell membranes) & soluble fractions by centrifugation

3. Phosphorylase was present only in supernatant (soluble fractions), but the response to hormone required the particulate fraction – experiments show that it is at least 2-step response

B. Incubate epinephrine or glucagon with broken cell preparation —> phosphorylase activated

C. Isolate particulate fraction, treat with hormone, wash & add wash to supernatant —> phosphorylase activated; wash contained substance that activated phosphorylase

1. Sutherland identified the substance released by the particulate fraction membranes as cyclic adenosine monophosphate (cyclic AMP or cAMP)

2. This substance (cAMP) was released by particulate fraction into wash & activated phosphorylase

3. cAMP —> glucose mobilization stimulated; activates a protein kinase that adds a phosphate group to a specific serine residue of the phosphorylase polypeptide —> activates phosphorylase

D. cAMP is 2nd messenger - released into cytoplasm due to 1st messenger (hormone or other ligand) binding at cell outer surface & diffuses to other sites in cell

1. While 1st messenger binds only to a single receptor species, 2nd messengers stimulate variety of cell activities leading to large-scale, coordinated response after stimulation by single extracellular ligand

2. A number of other 2nd messengers have been found in eukaryotic cells: Ca2+ ions, phosphoinositides, inositol triphosphate, diacylglycerol, cGMP & nitric oxide

Phosphatidylinositol-Derived Second Messengers – Background

I. Cell membrane phospholipids were originally viewed as structural components that make membranes cohesive & impermeable to aqueous solutes

A. It is now known that they form precursors of a number of 2nd messengers - phospholipids are converted into 2nd messengers by a variety of enzymes that are regulated in response to extracellular signals

B. The enzymes that convert phospholipids into 2nd messengers include:

1. Phospholipases (lipid-splitting enzymes)

2. Phospholipid kinases (lipid-phosphorylating enzymes)

3. Phospholipid phosphatases (lipid-dephosphorylating enzymes)

C. Phospholipases are enzymes that hydrolyze specific ester bonds that connect the different building blocks that make up a phospholipid molecule; there are 4 classes of these enzymes

1. They can be activated in response to extracellular signals

2. The products they produce function as second messengers

II. Best-studied lipid 2nd messengers derived from phosphatidylinositol & generated after transmission of signals by G protein-coupled receptors; another group of 2nd messengers is derived from sphingomyelin

Phosphatidylinositol-Derived Second Messengers – Phosphatidylinositol Phosphorylation

I. Examples of different responses (contraction & secretion) triggered by the same 2nd messenger, a substance derived from the compound phosphatidylinositol, a minor component of most cellular membranes

A. When the neurotransmitter acetylcholine binds to the surface of a smooth muscle cell within the wall of the stomach —> the muscle cell is stimulated to contract

B. When a foreign antigen binds to the surface of a mast cell, the cell is stimulated to secrete histamine, a substance that can trigger the symptoms of an allergy attack

II. First indication that phospholipids might be involved in cell responses to extracellular signals emerged from studies below

A. Lowell & Mabel Hokin (early 1950s; Montreal General Hospital & McGill Univ.) - they had set out to study acetylcholine effects on pancreatic mRNA synthesis

1. Incubate pigeon pancreas slices in 32PO4 (orthophosphate); this is the usual way of getting radiolabeled nucleotide triphosphates, which are used as precursors during RNA synthesis

2. Tissue treatment with acetylcholine led to radiolabel incorporation into cell phospholipid fraction (mostly PI); it was then quickly changed to other phosphorylated derivatives (phosphoinositides)

3. Suggested that lipids can be phosphorylated by specific lipid kinases that are activated in response to extracellular messenger molecules like acetylcholine

B. It is now well established that lipid kinases are activated in response to a large variety of extracellular signals

III. Reactions of PI metabolism – the inositol ring resides at the inner polar surface of bilayer & has 6 carbons

A. Carbon #1 is involved in joining inositol to diacylglycerol

B. The 3, 4 or 5 carbons can be phosphorylated by specific phosphoinositide kinases present in cells

1. Add single phosphate to PI inositol sugar with PI 4-kinase (PI4K) —> generates PI 4-phosphate (PIP)

2. PIP can be phosphorylated a second time with PIP 5-kinase (PIP5K) to form PI 4,5-bisphosphate (PIP2)

3. PIP2 can be phosphorylated again by PI 3-kinase (PI3K) to form PI 3,4,5-trisphosphate (PIP3);

4. PIP2 phosphorylation to form PIP3 is of particular interest since PI3K enzymes involved in process can be controlled by a large variety of extracellular molecules

5. Also PIP3 overactivity has been associated with human cancers (e.g., formed in insulin response)

6. All 3 of the phospholipid species discussed above remain in plasma membrane cytoplasmic leaflet

B. There are also lipid phosphatases to remove the phosphate groups added by the lipid kinases

C. The activities of these kinases & phosphatases are coordinated so that specific phosphoinositides appear at specific regions of the membrane at specific times after a signal has been received

IV. Phosphorylated inositol rings of phosphoinositides form binding sites for several lipid-binding domains found in proteins; best known are the PH domains, which have been identified in >150 different proteins

A. Protein binding to PIP2 or PIP3 2nd messengers recruits these proteins to cytoplasmic face of membrane, where they can interact with other membrane-bound proteins, like activators, inhibitors or substrates

B. Example: in cells involved in chemotaxis (raising concentration of a particular chemical in the medium that serves as a chemoattractant), PIP3 is specifically localized to a particular part of cell membrane

1. This mechanism causes phagocytic cells like macrophages to move toward bacteria or other targets that they engulf

2. Recent studies indicate that chemotaxis depends on the localized production of phosphoinositide messengers that act like a compass to inform the cell of the target location

V. Phospholipase C – not all inositol-containing 2nd messengers remain in the membrane's lipid bilayer

A. When acetylcholine binds to smooth muscle cell (in blood vessel or stomach wall) or foreign antigen binds to mast cell, the bound receptor activates a heterotrimeric G protein

1. Acetylycholine binding to smooth muscle cell causes contraction; foreign antigen binding to a mast cell causes histamine secretion

2. The heterotrimeric G protein, in turn, activates an effector, phosphatidylinositol (PI)-specific phospholipase C- (PLC)

3. PLC is situated at the inner surface of the membrane, bound there by the interaction between its PH domain & a phosphoinositide embedded in the bilayer

B. PLC (at inner membrane surface) catalyzes split of PIP2 into 2 molecules, both of which are second messengers important in cell signaling: inositol 1,4,5-triphosphate (IP3) & diacylglycerol (DAG)

1. Both of these molecules play important roles as 2nd messengers in cell signaling

VI. Diacyglycerol – a lipid molecule that remains in membrane after its formation by PLC

A. Recruits & activates an effector called protein kinase C (PKC), a family of related enzymes that phosphorylate serine & threonine residues on a wide variety of target proteins

1. PKC is multifunctional, serine & threonine kinase that acts on wide variety of target proteins

2. Has many important roles in cell growth/differentiation, cell metabolism & transcriptional activation

B. Example of PKC's importance in growth control: phorbol esters (powerful plant compounds that resemble DAG) activate PKC in a variety of cultured cells

1. Cells treated with phorbol esters lose growth control —> behave malignant temporarily

2. Remove phorbol esters from medium —> cells recover their normal growth properties

3. Application of phorbol esters to the skin in combination with certain other chemicals will cause the formation of skin tumors

C. Another example: genetically engineer cells to constitutively (continually) express protein kinase C —> they exhibit permanently malignant phenotype in cell culture & can cause tumors in susceptible mice

VII. Inositol 1,4,5-triphosphate (IP3) - small, water soluble sugar phosphate; capable of rapid diffusion throughout cell interior; the effect of IP3 is usually transient since it is rapidly inactivated enzymatically

A. IP3 forms at membrane, diffuses into cytosol & binds to a specific IP3 receptor at SER surface; SER is a site of Ca2+ storage in a variety of cells

B. IP3 receptor is also a tetrameric Ca2+ channel; IP3 binding opens channel & allows Ca2+ ions to diffuse from SER into cytoplasm; calcium ions can also be considered as intracellular or second messengers

1. Ca2+ ions bind to various target molecules, triggering specific responses

C. Examples: smooth muscle contraction & exocytosis of histamine-containing secretory vesicles; both are triggered by elevated calcium levels

D. Example: liver cell response to vasopressin (same hormone that causes antidiuretic activity in kidney)

1. Vasopressin binds to its receptor at liver cell surface & causes a series of IP3-mediated bursts of Ca2+ release that appear as oscillations of free cytosolic calcium concentration

2. The frequency & intensity of such oscillations may encode information that governs the cell's specific response

The Specificity of G Protein-Coupled Responses

I. Wide variety of agents (hormones, neurotransmitters, sensory stimuli) act by way of GPCRs & heterotrimeric G proteins to transmit information across plasma membrane; triggers a wide variety of cell responses

II. The various parts of the signal transduction machinery are not identical in every cell type

A. Receptors for a given ligand can exist in several different versions (isoforms)

1. Researchers have identified 9 different isoforms of the adrenergic receptor, which binds epinephrine

2. 15 different isoforms of the receptor for serotonin exist; serotonin is a powerful neurotransmitter released by nerve cells in parts of the brain governing emotions

3. Different isoforms can have different affinities for the ligand or may interact with different types of G proteins

4. Different receptor isoforms may coexist in the same plasma membrane or they may occur in the membranes of different types of target cells

B. The heterotrimeric G proteins that transmit signals from receptor to effector can also exist in multiple forms, as can many of the effectors

1. The human genome encodes at least 16 different G subunits, 5 different G subunits & 11 different G subunits identified, along with 9 isoforms of the effector adenylyl cyclase have also been identified

C. Different combinations of specific subunits construct G proteins having different capabilities of reacting with specific isoforms of both receptors & effectors

D. Some G proteins act by inhibiting their effectors; the same stimulus can activate a stimulatory G protein (with a Gs subunit) in one cell & an inhibitory G protein (with a Gi subunit) in a different cell

1. When epinephrine binds a -adrenergic receptor on cardiac muscle cell, a G protein with a Gs subunit is activated, stimulating cAMP production & leading to a rise in contraction rate & force

2. When epinephrine binds to an -adrenergic receptor on a smooth muscle in the intestine, a G protein with a Gi subunit is activated, inhibiting cAMP production & causing muscle relaxation

3. Some adrenergic receptors turn on G proteins with Gq subunits, leading to PLC activation

E. Thus, clearly the same extracellular messenger can activate a variety of pathways in different cells

Regulation of Blood Glucose Levels

I. Glucose can be utilized as a source of energy by all cell types present in the body

A. It is oxidized to CO2 & H2O by glycolysis & the Krebs (TCA) cycle

B. The ATP produced provides cells with ATP that can be used to drive energy-requiring reactions

II. Because it is such an important resource, the body maintains glucose levels in the bloodstream within a narrow range

A. Excess glucose in animal cells is stored as glycogen (a large, branched glucose polymer of glucose monomers linked together through glycosidic bonds)

B. The hormone glucagon is produced by the alpha () cells of the pancreas in response to low blood glucose levels

1. Glucagon stimulates the breakdown of glycogen & the release of glucose into the bloodstream

2. This causes bloodstream glucose levels to rise

C. The hormone insulin is produced by the beta () cells of the pancreas in response to high glucose levels & stimulates glucose uptake & storage as glycogen

1. Insulin acts through a receptor protein-tyrosine kinase

D. Epinephrine (sometimes called the "fight-or-flight" hormone) is produced by the adrenal gland in stressful situations

1. Epinephrine causes an increase in blood glucose levels to provide the body with the extra energy resources needed to deal with the stressful situation at hand

III. Binding of either epinephrine or glucagons initiates a series of reactions that leads to activation of the enzyme glycogen phosphorylase, which catalyzes the breakdown of glycogen into glucose 1-phosphate

A. In addition, binding of either of these hormones leads to inhibition of the enzyme glycogen synthase, which catalyzes the opposing reaction in which glucose units are added to growing glycogen molecules

B. Thus, two different stimuli (glucagon & epinephrine), recognized by different receptors, induce the same response in a single target cell

IV. The receptor for glucagon is a G protein-coupled receptor

A. Glucagon is a small protein that is composed of 29 amino acids, whereas epinephrine is a small molecule that is derived from the amino acid tyrosine

B. Structurally speaking, these 2 molecules have nothing in common, yet both of them stimulate the breakdown of glycogen into glucose 1-phosphate after binding to G protein-coupled receptors

C. The 2 receptors differ from one another primarily in the structure of the ligand-binding pocket on the extracellular surface of the cell, which is specific for one or the other hormone

D. After activation by their respective ligands, both receptors activate the same type of heterotrimeric G proteins that cause an increase in the levels of cAMP

V. Glucose mobilization: An example of a response induced by cAMP

A. Integral membrane protein adenylyl cyclase (catalytic domain resides at inner membrane surface) is effector (brings about cell response); makes cAMP, which starts reaction chain that mobilizes glucose

1. Receptor binds glucagon or epinephrine —> conformational shift; 1st step in reaction cascade

2. Change transmitted across membrane as the receptor activates a Gs subunit, which activates an adenylyl cyclase effector on the inner membrane surface

3. Activated adenylyl cyclase converts ATP to cAMP that rapidly diffuses to cytoplasm

B. Once formed, cAMP diffuses into the cytoplasm, where it binds to the allosteric site on the regulatory subunit of a cAMP-dependent protein kinase (protein kinase A; PKA)

1. PKA in its inactive form is a heterotetramer made of 2 regulatory (R) & 2 catalytic (C) subunits

2. The regulatory subunits normally inhibit the catalytic activity of the enzyme

3. cAMP binding causes the dissociation of the regulatory subunits, thereby releasing the catalytic subunits of PKA in their active form

C. The target substrates of PKA in a liver cell include 2 enzymes that play a pivotal role in glucose metabolism: glycogen synthase & phosphorylase kinase

1. Phosphorylation of glycogen synthase inhibits its catalytic activity & thus prevents the conversion of glucose to glycogen

2. Phosphorylation of phosphorylase kinase activates the enzyme to catalyze the transfer of phosphate groups to phosphorylase molecules

D. Krebs & Fischer discovered that the addition of a single phosphate group to a specific serine residue in the phosphorylase polypeptide activates the enzyme, stimulating the breakdown of glycogen

1. The glucose 1-phosphate formed in the phosphorylase reaction is converted to glucose, which diffuses into the bloodstream & so reaches the other tissues of the body

VI. A mechanism must exist to reverse the hormone's effect or the cell would remain in the activated state indefinitely - liver cells contain phosphatases that remove phosphate groups added by kinases

A. Protein phosphatase-1, a particular member of this family, removes PO43- from all of the phosphorylated enzymes: phosphorylase kinase, glycogen phosphorylase & glycogen synthase

B. cAMP phosphodiesterase helps to terminate the response to cAMP through the destruction of cAMP molecules present in the cell

C. Finally, it is important to stress that regulation of glucose metabolism is just one of many functions that is carried out by cAMP

VII. Signal amplification – a cascade greatly amplifies the signal generated from the original message; the hormone concentration in blood usually very low (<10-8 M)

A. A single hormone molecule bound at the cell surface activates a number of G proteins, each of which can activate an adenylyl cyclase effector

B. Each adenylyl cyclase produces many cAMPs in a short period of time

1. Thus, the production of a 2nd messenger provides a mechanism to greatly amplify the signal generated from the original message

2. Many steps in the reaction cascade result in amplification of the signal

C. Each cAMP molecule activates one PKA molecule

D. Each PKA catalytic subunit phosphorylates a large number of phosphorylase kinase molecules

E. Each phosphorylase kinase phosphorylates, in turn, an even larger number of glycogen phosphorylase molecules

F. Each glycogen phosphorylase molecule catalyzes, in turn, the formation of much larger number of glucose phosphates

G. Thus, what starts as a barely perceptible stimulus at the cell surface is rapidly transformed into a major mobilization of glucose within the cell

G Protein-Coupled Receptors: Other Aspects of cAMP Signal Transduction Pathways

I. A few PKAs also translocate into the nucleus where they phosphorylate key nuclear proteins; one such nuclear protein is a transcription factor called CREB (cAMP response element-binding protein)

A. Phosphorylated CREB binds as a dimer to sites on DNA containing a particular nucleotide sequence (TGACGTCA), known as the cAMP-response element (CRE)

1. Response elements are sites in the DNA where transcription factors bind & increase the rate of transcription initiation

2. CREs are found in the regulatory regions of genes that play a role in the response to cAMP

B. In liver cells, several gluconeogenesis enzymes are produced in response to cAMP (their genes contain nearby CREs); gluconeogenesis is a pathway by which glucose is formed from glycolysis intermediates

C. Thus, glucagon & epinephrine activate catabolic enzymes that break down glycogen for glucose & also promote synthesis of anabolic enzymes that synthesize glucose from smaller precursors

II. cAMP is made in response to a wide variety of different ligands (1st messengers) in many different cells & mediates varied responses; operates in mammals & invertebrates; each cell has different response

A. cAMP pathways are implicated in nervous system processes (learning, memory, drug addiction)

1. Chronic opiate use —> elevated adenylyl cyclase & PKA levels, which may be partially responsible for the physiological responses occurring during drug withdrawal

B. Another cyclic nucleotide, cyclic GMP (cGMP) also acts as a 2nd messenger in certain cells, as illustrated by the induced relaxation of smooth muscle cells; it also plays a key role in the vision-signaling pathway

C. cAMP response in a particular cell is determined by specific proteins acted on by PKA, since most of cAMP's effects are exerted through PKA; kinase substrates expressed in target cells differ cell to cell

1. In liver cell, PKA activation in response to epinephrine leads to glycogen breakdown

2. In kidney tubule cells, response to vasopressin causes an increase in membrane permeability to H2O

3. In thyroid cell, response to TSH leads to thyroid hormone secretion

D. Clearly, PKA must phosphorylate different substrates in each of these cell types, thereby linking the cAMP level increases induced by epinephrine, vasopressin & TSH to different physiological responses

1. >100 PKA substrates found; most carry out separate functions thus raising question about how PKA phosphorylates appropriate substrates in response to a particular stimulus in a particular cell type

2. Answered in part by the fact that different cells express different PKA substrates & in part by discovery of PKA-anchoring proteins or AKAPs that function as signaling hubs

E. The first AKAPs were discovered as proteins that co-purified with PKA; >30 AKAPs have been discovered since the first one

1. AKAPs provide a structural framework for coordinating protein-protein interactions by sequestering PKA to specific locations within the cell

2. As a consequence, PKA accumulates in close proximity to one or more substrates

3. When cAMP levels rise & PKA is activated, the relevant substrates are present close by & they are the first ones to become phosphorylated

4. Substrate selection thus is partly a consequence of PKA localization in the presence of particular substrates

5. Different cells express different AKAPs, resulting in localization of PKA in presence of different substrates & consequently phosphorylation of different substrates after an increase in cAMP levels

6. Interesting fact - unlike most proteins with similar function, AKAPs have diverse structure; suggests that evolution co-opted a variety of different protein types to carry out similar cell signaling role

G Protein-Coupled Receptors: The Role of G Protein-Coupled Receptors in Sensory Perception

I. The ability to see, taste & smell depends largely on GPCRs; rhodopsin is an example of G protein function

II. Rhodopsin is the light-sensitive protein found in retina rods, which are photoreceptor cells that respond to low light intensity & give us a black-&-white picture of our environment at night or in a darkened room

A. Several closely related GPCRs are found in retina cones, which give us color vision in brighter light

B. Absorption of a single light photon by rhodopsin induces a conformational change in the protein, which transmits a signal to a heterotrimeric G protein (transducin) &, in turn, activates a coupled effector

C. The effector is the enzyme cGMP phosphodiesterase that hydrolyzes the cyclic nucleotide, cGMP

D. cGMP hydrolysis triggers the closure of certain cation-specific channels, leading to the generation of membrane potentials that may be transmitted as action potentials along the optic nerve

III. Our sense of smell depends on nerve impulses transmitted along olfactory neurons that extend from the epithelia lining nasal cavities to the olfactory bulb in the brainstem

A. Distal tips of these neurons located in the nasal epithelium contain odorant receptors (GPCRs that are able to bind various chemicals that enter our nose)

1. Mammalian odorant receptors were first identified in 1991 by Linda Buck & Richard Axel (Columbia Univ.)

2. It is estimated that humans express roughly 400 different odorant receptors that, taken together, can combine with a large variety of different chemical structures (odorants)

3. The human genome contains roughly 1000 genes that encode odorant receptors, but the majority is present as nonfunctional pseudogenes

4. Mice, who depend more heavily than humans on their sense of smell, have >1000 of these genes in their genome, & 95% of them encode functional receptors

5. Each olfactory neuron is thought to contain only one of the hundreds of different odorant receptors encoded by the genome & thus can only respond to one or a few related chemicals

6. Thus, activation of different neurons containing different odorant receptors provides us with the perception of different aromas

B. Mutations in a specific gene encoding a particular odorant receptor can leave a person with an inability to detect a particular chemical in the environment that most other members of population can perceive

C. When activated by bound ligands, odorant receptors signal through heterotrimeric G proteins to adenylyl cyclase —> results in cAMP synthesis —> opening of a cAMP-gated cation channel

1. This response ultimately leads to the generation of action potentials that are transmitted to the brain

IV. Taste perception operates at a much cruder level that the perception of smell, thus taste is much less discriminating than the perception of smell

A. Each taste receptor cell in tongue transmits a sense of one of only four basic taste qualities: salty, sour, sweet or bitter

1. A 5th type of taste receptor cell responds to monosodium glutamate or disodium guanylate, which are commonly added to processed foods to enhance flavor

2. Perception of food/beverage as salty or sour is elicited directly by Na ions or protons in food

3. It is proposed that these ions enter cation channels in taste receptor cell plasma membrane, leading to membrane depolarization

4. In contrast, the perception that a food is sweet or bitter depends on a compound interacting with a GPCR at the receptor cell surface

B. Recent studies in 2000 have provided information on perception of bitter-tasting substances

1. Humans encode a family of ~25 bitter-taste receptors (T2Rs), which are coupled to the same heterotrimeric G protein

2. These receptors bind a diverse group of different compounds, like plant alkaloids or cyanides that taste bitter

3. Most substances that evoke this perception are toxic compounds that elicit a distasteful, protective response that causes us to expel the food from our mouth

4. Unlike olfactory cells with a single receptor protein, a single taste-bud cell that evokes a bitter sensation has a wide variety of different T2R receptors that respond to unrelated noxious substances

5. Thus, many diverse substances evoke the same basic bitter & disagreeable taste

C. In contrast, a food eliciting a sweet taste is likely to be one that contains energy-rich carbohydrates

1. To date, studies suggest that humans possess only one sweet-taste GPCR (a T1R2 – T1R3 heterodimer) & it responds to both sugars & artificial sweeteners

D. Fortunately, food that is chewed releases odorants that travel via the throat to olfactory receptor cells in nasal mucosa

1. This allows the brain to learn much more about the food we have eaten than the simple messages provided by taste receptors

2. Thus, merged input from gustatory (taste) & olfactory neurons gives us our rich sense of taste

3. The importance of olfactory neurons in taste perception is more evident if we have a cold that causes us to lose some of our appreciation for the taste of food

Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction

I. Protein-tyrosine kinases are enzymes that phosphorylate specific tyrosine residues on protein substrates

A. Protein-tyrosine phosphorylation is mechanism for signal transduction that appeared with the evolution of multicellular organisms; >90 different protein-tyrosine kinases are encoded by the human genome

B. These kinases are involved in the regulation of cell growth, cell division, cell differentiation, cell survival, attachment to the extracellular matrix & migration

1. Expression of mutant protein-tyrosine kinases that cannot be regulated & are continually active can lead to uncontrolled cell division & the development of cancer

2. One type of leukemia occurs in cells that contain an unregulated version of the protein-tyrosine kinase ABL

II. Protein-tyrosine kinases can be divided into 2 groups: receptor protein-tyrosine kinases (RTKs) & non-receptor or cytoplasmic protein-tyrosine kinases

A. RTKs – integral membrane proteins containing an extracellular ligand-binding domain

1. Activated directly by extracellular growth & differentiation factors – epidermal growth factor (EGF) & platelet derived growth factor (PDGF) or by metabolic regulators like insulin

B. Nonreceptor protein-tyrosine kinases – regulated indirectly by extracellular signals & they control processes as diverse as the immune response, cell adhesion & neuronal cell migration

III. Receptor dimerization – it is widely accepted that ligand binding to protein-tyrosine kinases causes the dimerization of the extracellular ligand-binding domains of a pair of receptors – 2 mechanisms recognized

A. Ligand-mediated dimerization – early work suggested that ligands of RTKs have 2 receptor-binding sites, making it possible for one growth or differentiation factor to bind to 2 receptors at the same time

1. Thus, the ligand connects two receptors & causes ligand-mediated dimerization

2. Model is supported by the observation that growth & differentiation factors like PDGF or colony-stimulating factor-1 (CSF-1) are composed of 2 similar or identical disulfide-linked subunits

3. Each of the subunits of the growth or differentiation factor contains a receptor-binding site

B. Receptor-mediated dimerization – more recently, it was established that some growth factors contain only a single receptor-binding site

1. Structural work supports a second mechanism in which ligand binding induces a conformational change in the extracellular domain of a receptor

2. Conformational change leads to the formation or exposure of a receptor dimerization interface

3. The proposal is that the ligands act as allosteric regulators that turn on the ability of their receptors to form dimers

C. Regardless of the mechanism, receptor dimerization results in the juxtapositioning of 2 protein-tyrosine kinase domains on the cytoplasmic side of the plasma membrane

1. This brings 2 kinase domains into close contact allowing for trans-autophosphorylation

2. Thus, the protein kinase activity of one receptor of the dimer phosphorylates the tyrosine residues in the cytoplasmic domain of the other receptor of the dimer, and vice versa

IV. Autophosphorylation sites on RTKs can carry out 2 different functions: they can regulate kinase activity or serve as binding sites for cytoplasmic signaling molecules

A. Kinase activity is usually controlled by autophosphorylation on tyrosine residues that are present in the activation loop of the kinase domain

1. The activation loop, when unphosphorylated, obstructs the substrate-binding site, thereby preventing ATP from entering

2. After its phosphorylation, the activation loop is stabilized in a position away from the substrate-binding site, resulting in activation of the kinase domain

3. Once their kinase domain has been activated, the receptor subunits proceed to phosphorylate each other on tyrosine residues that are present in regions adjacent to the kinase domain

B. It is these autophosphorylation sites that act as binding sites for cellular signaling proteins

V. Phosphotyrosine-dependent protein-protein interactions – signaling pathways consist of a chain of signaling proteins that interact with one another in a sequential manner

A. Signaling proteins can associate with activated protein-tyrosine kinase receptors, since they contain domains that bind specifically to phosphorylated tyrosines – 2 such domains have been identified

1. The Src-homology 2 (SH2) domain

2. The phosphotyrosine-binding (PTB) domain

B. SH2 domains were initially identified as part of protein-tyrosine kinases encoded by the genome of tumor-causing (oncogenic) viruses

1. They are composed of ~100 amino acids & contain a conserved binding-pocket that accommodates a phosphorylated tyrosine residue

2. >110 SH2 domains are encoded by the human genome & they mediate a large number of phosphorylation-dependent protein-protein interactions

3. These interactions occur after phosphorylation of specific tyrosine residues

4. The specificity of the interactions is determined by the amino acid sequence immediately adjacent to the phosphorylated tyrosine residues

5. For example, the SH2 domain of the Src protein-tyrosine kinase recognizes P.Tyr-Glu-Glu-Ile, whereas the SH2 domains of PI 3-kinase bind to P.Tyr-Met-X-Met (in which X can be any residue)

6. Interestingly, the budding-yeast genome encodes only one SH2-domain-containing protein; this correlates with the overall lack of tyrosine kinase signaling activity in these lower eukaryotes

C. PTB domains were discovered more recently – they can bind to phosphorylated tyrosine residues that are usually present as part of an asparagine-proline-X-tyrosine (Asn-Pro-X-Tyr) motif

1. Some PTB domains appear to bind specifically to an unphosphorylated Asn-Pro-X-Tyr motif, whereas others bind to the phosphorylated motif

2. PTB domains are poorly conserved & different PTB domains possess different residues that interact with their ligands

VI. Downstream signaling pathway activation – receptor activation —> signaling complex formation with SH2- or PTB-containing signaling proteins (several groups) binding to specific receptor autophosphorylation sites

A. Adaptor proteins – function as linkers enabling ≥2 signaling proteins to be joined together as part of a signaling complex; they contain an SH2 domain & ≥1 additional protein-protein interaction domains

1. Ex.: adaptor protein Grb2 has 1 SH2 & 2 SH3 (Src-homology 3) domains; SH3 domains bind to a proline-rich sequence motif; Grb2 SH3 domains bind constitutively to other proteins (like Sos & Gab)

2. The SH2 domain binds to phosphorylated tyrosine residues within a Tyr-X-Asn motif

3. Thus, tyrosine phosphorylation of Sos or Gab Tyr-X-Asn motif results in translocation of Grb2-Sos or Grb2-Gab from the cytoplasm to the receptor, which is present at the plasma membrane

B. Docking proteins – supply certain receptors with additional tyrosine phosphorylation sites

1. They contain either a PTB domain or an SH2 domain & a number of tyrosine phosphorylation sites

2. Binding of extracellular ligand to receptor leads to autophosphorylation of receptor, providing a binding site for the PTB or SH2 domain of the docking protein

3. Once bound together, the receptor phosphorylates tyrosine residues found on the docking proteins

4. The docking protein phosphorylation sites then act as binding sites for more signaling molecules

5. They provide versatility to the signaling process, since the ability of the receptor to turn on signaling molecules can vary with the docking proteins that are expressed in a particular cell

C. Transcription factors – transcription factors that belong to the STAT family play an important role in the function of the immune system

1. STATs contain an SH2 domain together with a tyrosine phosphorylation site that can act as a binding site for its own SH2 domain

2. Tyrosine phosphorylation of STAT SH2 binding sites situated within a dimerized receptor leads to the recruitment of 2 STAT proteins

3. Upon association with the receptor complex, tyrosine residues in these STAT proteins are phosphorylated

4. Due to the interaction between the phosphorylated tyrosine residue on one STAT protein & the SH2 domain on the second STAT protein & vice versa, the transcription factors form dimers

5. Dimers, but not monomers, move to the nucleus where they stimulate the transcription of specific genes involved in an immune response

D. Signaling enzymes – protein & lipid kinases, protein phosphatases, phospholipases, GTPase-activating proteins; have SH2 domains, bind active RTKs; turned on directly or indirectly by 3 mechanisms:

1. They may be activated simply as a result of translocation to the membrane, which places them in close proximity to their substrates

2. They may also be activated by an allosteric mechanism in which phosphotyrosine binding causes SH2 domain conformational change & then resulting changes in catalytic domain shape & activity

3. Finally, enzymes can be regulated directly by phosphorylation

E. In summary, signaling proteins that associate with activated RTKs initiate cascades of events that lead to the biochemical changes required to respond to the presence of extracellular messenger molecules

VII. Ending the response – signal transduction by RTKs is usually terminated by receptor internalization; what causes this to happen is still an area of active research; example – receptor-binding protein named Cbl

A. When RTKs are activated by ligands, they autophosphorylate tyrosine residues, which can act as a binding site for Cbl

B. Cbl then associates with the receptor & catalyzes the attachment of a ubiquitin molecule to the receptor

1. Ubiquitin is a small protein that is linked covalently to other proteins, thereby marking those proteins for internalization or degradation

C. Cbl complex binding to activated receptors is followed by receptor ubiquitination, internalization &, in most cases, degradation in a lysosome

RTK-Activated Signaling Pathways: The Ras-MAP Kinase Pathway

I. Retroviruses are small viruses that carry their genetic information in the form of RNA – some have genes called oncogenes that enable them to transform normal cells into tumor cells

II. Ras - originally described as retroviral oncogene; eventually, found that retroviral Ras gene was derived from previously infected mammalian host; ~30% of all human cancers contain Ras gene mutant versions

A. Important to note that Ras proteins are part of a superfamily of >100 small G proteins including the Rabs, Arf1 & Ran

1. These proteins are involved in regulation of numerous processes, including gene expression, cell division, nucleocytoplasmic transport, differentiation, cytoskeletal organization & vesicle trafficking

2. Principles discussed in connection with Ras apply to many members of the small G-protein superfamily

B. Ras is a small GTPase (G protein) that is held at the inner surface of the plasma membrane by a lipid group that is embedded in the inner leaflet of the bilayer

1. Ras has a function similar to that of heterotrimeric G proteins discussed above; Ras acts as both a switch & a molecular timer

2. Unlike heterotrimeric G proteins, however, Ras consists of only a single small subunit

C. Ras proteins are present in 2 different forms: an active GTP-bound form & an inactive GDP-bound form

1. Ras-GTP binds & activates downstream signaling proteins

2. Ras is turned off by hydrolysis of its bound GTP to GDP

D. Mutations in the RAS gene that lead to tumor formation prevent the protein from hydrolyzing the bound GTP back to the GDP form; the mutant version of "Ras" stays in the "on" position

1. The mutant thus sends a continuous signal downstream along the signaling pathway, keeping the cell in the proliferative mode

III. Cycling of monomeric G proteins like Ras between active & inactive states is aided by accessory proteins that bind to the G protein & regulate its activity; these accessory proteins include:

A. GTPase-activating proteins (GAPs) – most monomeric G proteins possess some capability to hydrolyze a bound GTP, but this capability is greatly accelerated by interaction with specific GAPs

1. Since they stimulate hydrolysis of the bound GTP, which inactivates the G protein, GAPs dramatically shorten the duration of a G protein-mediated response

2. Mutations in one of the Ras-GAP genes (NF1) cause neurofibromatosis 1, a disease in which patients develop large numbers of benign tumors (neurofibromas) along the sheaths lining the nerve trunks

B. Guanine nucleotide-exchange factors (GEFs) – an inactive G protein is converted to the active form when the bound GDP is replaced with a GTP

1. GEFs are proteins that bind to an inactive monomeric G protein & stimulate dissociation of the bound GDP

2. Once the GDP is released, the G protein rapidly binds a GTP, which is present at relatively high concentration in the cell —> the G protein is activated

C. Guanine nucleotide-dissociation inhibitors (GDIs) – GDIs are proteins that inhibit the release of a bound GDP from a monomeric G protein, thus maintaining the protein in the inactive, GDP-bound state

IV. Ras-GTP is thought to interact directly with several downstream targets - one role is as an element of Ras-MAP kinase cascade

A. The Ras-MAP kinase cascade is turned on in response to a wide variety of extracellular signals & plays a key role in regulating vital activities like cell proliferation & differentiation

1. The pathway relays extracellular signals from plasma membrane through the cytoplasm & into the nucleus & is activated when a growth factor (EGF or PDGF) binds to its RTK's extracellular domain

B. Many activated RTKs have phosphorylated tyrosine residues that act as docking sites for the adaptor protein Grb2, which, in turn, binds to Sos, a guanine nucleotide-exchange factor (GEF) for Ras

1. Creation of Grb2 binding-site on activated receptor promotes translocation of Grb2-Sos from the cytoplasm to the cytoplasmic surface of plasma membrane, placing Sos in close proximity to Ras

C. Simply bringing Sos to plasma membrane is sufficient to cause Ras activation – illustrated by experiment with a mutant version of Sos that is permanently tethered to the plasma membrane inner surface

1. Expression of this membrane-bound Sos mutant results in constitutive activation of Ras & transformation of the cell to a malignant phenotype

D. Interaction with Sos opens the Ras nucleotide-binding site —> GDP is released & replaced by GTP

E. Exchange of GDP for GTP in the Ras nucleotide-binding site results in a conformational change resulting in the creation of a binding interface for an important signaling protein called Raf

F. Raf is then recruited to the inner surface of the plasma membrane where it is activated; Raf is a serine-threonine protein kinase, one of whose substrates is the protein kinase MEK

1. Raf activation requires its phosphorylation by several protein kinases & involves several protein-protein interactions that are regulated by these phosphorylation events

2. The precise mechanism of Raf activation remains to be established

G. MEK is activated as a consequence of phosphorylation by Raf & goes on to phosphorylate & activate 2 MAP kinases (Erk-1 & Erk-2)

1. >160 proteins that can be phosphorylated by these kinases have been identified

2. These proteins include transcription factors, protein kinases, cytoskeletal proteins, apoptotic regulators, receptors & other signaling proteins

H. Once activated, the MAP kinase is able to move into the nucleus where it phosphorylates & activates specific transcription factors, such as Elk-1, Fos & Jun

1. Eventually, the pathway leads to the activation of genes involved in cell proliferation, including cyclin D1, which plays a key role in driving a cell from G1 into S phase

V. Oncogenes are identified by their ability to cause cells to become cancerous; they are derived from normal cellular genes that have either become mutated or are overexpressed

A. Many of the proteins that play a role in the Ras signaling pathway were discovered because they are encoded by cancer-causing oncogenes

1. This includes the genes for Ras, Raf & a number of the transcriptional factors activated at the end of the pathway (e.g., Fos & Jun)

B. Genes for several of the RTKs situated at the beginning of the pathway, including receptors for both EGF & PDGF have also been identified among the several dozen known oncogenes

C. The fact that so many proteins in this pathway are encoded by genes that can cause cancer when mutated emphasizes the importance of the pathway to the control of cell growth & proliferation

VI. MAP kinase cascade transmits different types of information - same basic pathway (RTKs —> Ras & transcription factor activation is found in all eukaryotes studied (yeasts, flies, nematodes, mammals)

A. Evolution has adapted the pathway to meet many different ends

1. Yeast – MAP kinase cascade is required for cells to respond to mating pheromones

2. Fruit flies – pathway is utilized during the differentiation of the photoreceptors in the compound eye

3. Flowering plants – pathway transmits signals that initiate a defense against pathogens

B. In each case, the pathway core contains 3 enzymes that act sequentially: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) & a MAP kinase (MAPK)

1. Each of these components is represented by a small family of proteins

2. 14 different MAPKKKs, 7 different MAPKKs & 13 different MAPKs identified in mammals

3. By utilizing different members of these protein families, mammals can assemble a number of different MAP kinase pathways that transmit different types of extracellular signals

C. In cells exposed to stressful stimuli (X-rays, damaging chemicals), signals are transmitted along different MAP kinase pathways that cause cell withdrawal from the cell cycle, rather than passing through it

1. Contrasts with the previously described MAP kinase cascade leading to cell proliferation

2. Withdrawal from cell cycle gives the cell time to repair damage resulting from adverse conditions

D. Recent studies have focused on the specificity of MAP kinase cascades in an attempt to understand how cells can use similar proteins as components of pathways that elicit different cellular responses

1. Studies of amino acid sequences & protein structure suggest that part of the answer lies in selective interactions between enzymes & substrates

2. Certain MAPKKK family members phosphorylate specific members of the MAPKK family, which, in turn phosphorylate specific members of the MAPK family

3. But many members of these families can participate in more than one MAPK signaling pathway

E. Specificity in MAP kinase pathways is also achieved by spatial localization of the component proteins

1. Spatial localization is done by structural (i.e., nonenzymatic) proteins called scaffolding proteins

2. Their apparent function is to tether appropriate members of a signaling pathway in a specific spatial orientation that enhances their mutual interactions

3. Scaffolding proteins may also take an active role in signaling events by inducing a change in conformation of bound proteins

4. In addition to facilitating a particular series of reactions, scaffolding proteins may prevent proteins involved in one signaling pathway from participating in other pathways

RTK-Activated Signaling Pathways: Signaling by the Insulin Receptor

I. Our bodies spend considerable effort maintaining blood-glucose levels within a narrow range

A. Effects of abnormal blood-glucose levels

1. A decrease in blood-glucose levels can lead to a loss of consciousness & coma, since the central nervous system depends largely on glucose for its energy metabolism

2. A persistent elevation in blood-glucose levels results in a loss of glucose, fluids & electrolytes in the urine & serious health problems

B. The levels of glucose in the circulation are monitored by the pancreas

1. When blood glucose levels fall below a certain level, pancreas cells secrete glucagon; glucagon acts through GPCRs & stimulates glycogen breakdown resulting in blood glucose level increase

2. When glucose levels rise, as occurs after a carbohydrate-rich meal, the cells of the pancreas respond by secreting insulin

3. Insulin functions as an extracellular messenger molecule, informing cells that glucose levels are high

4. Cells that express insulin receptors on their surface (e.g., liver cells) respond to insulin by increasing glucose uptake, increasing glycogen & triglyceride synthesis & decreasing gluconeogenesis

II. The insulin receptor is a protein-tyrosine kinase - each insulin receptor is composed of an & a chain, which are derived from a single precursor protein by proteolytic processing

A. The chain is entirely extracellular & contains the insulin-binding site; the chain is composed of an extracellular region, a single transmembrane region & a cytoplasmic region

1. The & chains are linked together by disulfide bonds

2. Two of these heterodimers are held together by disulfide bonds between the chains

B. While most RTKs are present on the cell surface as monomers, insulin receptors are present as stable dimers; like other RTKs, insulin receptors are inactive in the absence of ligand

1. Recent work suggests that the insulin receptor dimer binds a single insulin molecule

2. Insulin binding causes a repositioning of the ligand-binding domains on the cell exterior, which causes the tyrosine kinase domains on the inside of the cell to come into close physical proximity

3. Juxtaposition of the kinase domains leads to autophosphorylation & receptor activation

III. Several tyrosine phosphorylation sites have been identified in the insulin receptor cytoplasmic region – 3 of these phosphorylation sites are present in the activation loop

A. In the unphosphorylated state, the activation loop assumes a conformation in which it occupies the active site

B. Upon phosphorylation of the 3 tyrosine residues, the activation loop assumes a new conformation away from the catalytic cleft

1. This new conformation requires a rotation of the small & large lobes of the kinase domain with respect to each other, thereby bringing residues that are essential for catalysis closer together

2. In addition, the activation loop now leaves the catalytic cleft open so that it can bind substrates

3. After activation of the kinase domain, the receptor phosphorylates itself on tyrosine residues that are present adjacent to the membrane & in the carboxyterminal tail

IV. Insulin receptor substrates 1 and 2 – most RTKs possess autophosphorylation sites that directly recruit SH2 domain-containing signaling proteins; the insulin receptor is an exception to this general rule

A. The insulin receptor associates instead with a small family of docking proteins called insulin-receptor substrates (IRSs); IRSs, in turn, provide the binding sites for SH2 domain-containing signaling proteins

1. After ligand binding & kinase activation, the receptor autophosphorylates tyrosine 960, which then forms a binding site for the phosphotyrosine binding (PTB) domains of insulin receptor substrates

B. IRSs are characterized by the presence of an N-terminal PH domain, a PTB domain & a long tail containing tyrosine phosphorylation sites

1. The PH domain may interact with phospholipids present at the inside leaflet of the plasma membrane

2. The PTB domain binds to tyrosine phosphorylation sites on the activated receptor

3. Tyrosine phosphorylation sites provide docking sites for SH2 domain-containing signaling proteins

C. At least 4 members of the IRS family have been identified – based on results obtained in knock-out experiments in mice, it is thought that IRS-1 & IRS-2 are most relevant to insulin-receptor signaling

1. Autophosphorylation of the activated insulin receptor at tyrosine 960 provides a binding site for IRS-1 & IRS-2

2. Only after stable association with either IRS-1 or IRS-2 is the activated insulin receptor able to phosphorylate tyrosine residues present on these docking proteins

3. Both IRS-1 & IRS-2 contain a large number of potential tyrosine phosphorylation sites

4. Those identified for sure form binding sites for SH2 domains of PI 3-kinase, Grb2 & Shp2; these proteins associate with receptor-bound IRS-1 or IRS-2 & activate downstream signaling pathways

D. PI 3-kinase (PI3K) is made of 2 subunits, one containing 2 SH2 domains & the other containing the catalytic domain

1. PI3K, which is activated directly as a consequence of binding of its 2 SH2 domains to tyrosine phosphorylation sites, phosphorylates phosphoinositides at the 3-position of the inositol ring

2. The products of this enzyme, which include PI 3,4-bisphosphate (PIP2) & PI 3,4,5-trisphosphate (PIP3), remain in the cytosolic leaflet of the plasma membrane

3. In the plasma membrane's cytosolic leaflet, PIP2 & PIP3 provide binding sites for PH domain-containing signaling proteins, such as the serine-threonine kinases PKB & PDK1

4. PKB (also known as Akt) plays a key role in mediating the response to insulin, as well as to other extracellular signals

5. Recruitment of PDK1 to the plasma membrane, in close proximity to PKB, provides a setting in which PDK1 can phosphorylate & activate PKB

6. While phosphorylation by PDK1 is essential, it is not sufficient for activation of PKB; PKB activation also depends on phosphorylation by a second kinase, most likely mTor

V. Glucose transport – PKB is directly involved in regulating glucose transport & glycogen synthesis; however, the pathway between PKB & GLUT4 translocation remains to be defined

A. The glucose transporter GLUT4 carries out insulin-dependent glucose transport

1. In the absence of insulin, GLUT4 is present in membrane vesicles that are present in the cytoplasm of insulin-responsive cells

2. These vesicles fuse with the plasma membrane in response to insulin, a process that is referred to as GLUT4 translocation

3. The increase in numbers of glucose transporters in the plasma membrane leads to increased glucose uptake

4. GLUT4 translocation depends on PI 3-kinase & PKB activation, a conclusion based on experiments showing that inhibitors of PI3K block GLUT4 translocation

5. In addition, expression of activated PI3K or PKB mutants stimulates GLUT4 translocation

6. It is well known that many receptors turn on PI3K, whereas it is only the insulin receptor that stimulates GLUT4 translocation

7. This suggests that there is a second pathway downstream of the insulin receptor that is essential for GLUT4 translocation to occur

8. Detailed understanding of how the 2 pathways work together to stimulate GLUT4 translocation is still lacking

B. Excess glucose taken up by muscle & liver cells is stored as glycogen, the synthesis of which is catalyzed by glycogen synthase, an enzyme turned off by phosphorylation on serine & threonine residues

1. Glycogen synthase kinase-3 (GSK-3) has been identified as glycogen synthase's negative regulator

2. GSK-3, in turn, is inactivated after phosphorylation by PKB

3. Thus, activation of the PI 3-kinase-PKB pathway in response to insulin leads to a decrease in GSK-3 kinase activity, resulting in an increase in glycogen synthase activity

4. Activation of protein phosphatase 1, an enzyme known to dephosphorylate glycogen synthase, contributes further to glycogen synthase activation

RTK-Activated Signaling Pathways: The Insulin Receptor and Diabetes Mellitus

I. Diabetes mellitus – caused by defects in insulin signaling; comes in 2 varieties: type I (accounts for 5 – 10% of the cases) & type II (accounts for the remaining 90 – 95%)

A. Type I diabetes – caused by an inability to produce insulin

B. Type II diabetes – more complex disease whose incidence is increasing around the world at an alarming rate; the increase is most likely the result of changing lifestyle & eating habits

1. A high-calorie diet combined with a sedentary lifestyle is thought to lead to a chronic increase in insulin secretion

2. Elevated levels of insulin overstimulate target cells in the liver & elsewhere in the body, leading to a condition called insulin resistance (target cells stop responding to the presence of the hormone)

3. Supported by genetic experiments in mice – mutations in genes for either the insulin receptor or IRS-2 render the cells unable to respond to insulin & creates a diabetic phenotype

4. Mutations in these genes are rare in the human population & the exact basis of insulin resistance remains to be established

II. Regardless of the mechanism leading to insulin resistance, it results in elevated blood-glucose level since body cells cannot remove sufficient sugar from the blood

A. Elevated blood sugar stimulates additional secretion of insulin from the pancreas as the body attempts to increase glucose uptake by peripheral tissues

1. The vicious cycle of peripheral insulin resistance & elevated insulin secretion can lead in many cases to the destruction of the cells of the pancreas

2. One strategy for diabetes treatment is to prevent insulin resistance, by making the cells more insulin sensitive

B. Mice lacking negative regulators of insulin receptor signaling (protein tyrosine phosphatase 1B [PTP-1B]) have a marked increase in insulin sensitivity

1. This suggests that proteins active in the insulin signal transduction pathway can be used as targets for anti-diabetic drugs

2. PTP-1B is a phosphatase thought to remove phosphate groups from tyrosine residues on the insulin receptor, thereby inactivating the receptor & stopping the hormone response

3. Normally, mice that are fed high-fat diets develop insulin resistance characteristic of type II diabetes & they also become obese

4. In contrast, mice lacking PTP-1B (PTP-1B knockout mice) that are fed a high-fat diet display enhanced insulin sensitivity & maintain a normal blood-glucose level & body weight

5. The absence of PTP-1B phosphatase in these knockout mice is presumed to interfere with receptor inactivation, thereby increasing the animals' sensitivity to insulin

6. This suggests a treatment for diabetes through the use of drugs that specifically inhibit the human version of PTP-1B (being investigated as a drug target for treatment of diabetes & obesity)

Signaling Pathways in Plants

I. Plants & animals share certain basic signaling mechanisms, including the use of Ca2+ & phosphoinositide messengers; however, certain pathways are unique to each major kingdom

A. Cyclic nucleotides, probably the most ubiquitous animal cell messengers, seem to have little or no role in plant cell signaling

B. RTKs are also lacking in plant cells

C. However, plants have a type of protein kinase (a protein histidine kinase) that is absent from animal cells

II. In bacteria, a protein kinase that phosphorylates histidine residues & mediates the cell's response to a variety of environmental signals has been known for a long time

A. Until 1993, these protein kinases were thought to be restricted to bacteria, but flowering plants & yeasts were found in that year to have a protein kinase that phosphorylates histidines

1. In both eukaryotes, histidine protein kinases are transmembrane proteins: extracellular receptor domain for external stimuli + cytoplasmic histidine kinase domain that transmits signal to cytoplasm

2. Etr1 gene product is one of the best-studied plant histidine protein kinases

B. The Etr1 gene product is a receptor for the gas ethylene (C2H4), a plant hormone that regulates a diverse array of developmental processes, like seed germination, flowering & fruit ripening

1. Ethylene binding to its receptor leads to transmission of signals along a pathway that is very similar to the MAP kinase cascade found in animal cells & yeast

2. As in other eukaryotes, downstream targets of the MAP kinase pathway in plants are TFs that activate expression of specific genes encoding proteins required for the hormone response

3. As data obtained from sequencing Arabidopsis & other plant genomes is analyzed, similarities & differences between plant & animal signaling pathways should become more apparent

The Role of Calcium as an Intracellular Messenger: Introduction

I. Ca2+ ions play key role in remarkable variety of cell activities: muscle contraction, cell division, secretion (exocytosis), fertilization, synaptic transmission, metabolism, transcription, cell movement & cell death

II. In each of the above cases, an extracellular message is received at the cell surface & leads to a dramatic increase in concentration of Ca2+ ions within the cytosol

A. Ca2+ ion concentration in a particular cellular compartment is controlled by the regulated activity of Ca2+ ion pumps & channels located within the membranes surrounding the compartment

B. [Ca2+ ion] in cytosol of resting cell is maintained at very low levels - typically ~10-7 M

C. [Ca2+ ion] is >10,000x higher in the extracellular space or within the ER lumen or the plant cell vacuole than in cytosol; when it enters the cytoplasm, Ca2+ ions trigger a number of responses

D. Mitochondria also play an important role in sequestering & releasing Ca2+ ions, but their role is not as well understood & will not be discussed

III. Cytosolic Ca2+ level is kept very low because:

A. The Ca2+ ion channels in both the plasma & ER membranes are normally kept closed, making these membranes highly impermeable to this ion and

B. The energy-driven Ca2+ transport systems of the plasma & ER membranes normally pump calcium out of the cytosol

IV. Also abnormal elevation of cytosolic Ca2+ concentration, as can occur in brain cells following a stroke, can lead to massive cell death

The Role of Calcium as an Intracellular Messenger: IP3 and Voltage-Gated Ca2+ Channels

I. Many stimuli trigger a sudden increase in cytoplasmic Ca2+ ions by opening ion channels: fertilizing sperm, nerve impulses arriving at muscle cell, etc. through IP3 or other signals

A. Interaction of extracellular messenger molecule with a GPCR can lead to the activation of the enzyme phospholipase C-

1. Phospholipase C- splits the phosphoinositide PIP2 to release IP3, which opens calcium channels in the ER membrane, leading to a rise in cytosolic [Ca2+ ion]

B. Extracellular messengers that signal through RTKs can trigger a similar response

1. Primary difference - RTKs activate members of phospholipase C- subfamily, which possess an SH2 domain allowing their binding to activated, phosphorylated RTK, IP3 & voltage-gated Ca2+ channel

C. Another major route causes cytosolic [Ca2+ ion] rise

1. Nerve impulse —> plasma membrane depolarization —> triggers opening of plasma membrane voltage-gated Ca2+ channels —> Ca2+ ion influx from extracellular medium

II. There are 2 additional PLC isoforms: PLC (activated by Ca2+ ions) & PLC (activated by Ras-GTP)

A. All 4 PLC isoforms carry out the same reaction, producing IP3 & linking a multitude of cell surface receptors to an increase in cytoplasmic Ca2+

The Role of Calcium as an Intracellular Messenger: Visualizing Cytoplasmic Ca2+ Concentration in Real Time

I. Understanding of the role of Ca2+ ion in cellular responses has been greatly advanced by the development of indicator molecules that emit light in the presence of free calcium

A. In mid-1980s, new types of highly sensitive, fluorescent calcium-binding compounds (fura-2) were developed; they are synthesized in a form that can enter a cell by diffusing across its plasma membrane

1. Once inside the cell, the compound is modified to a form that cannot leave the cell

2. Using the probes, free Ca2+ ion concentration in different parts of living cell can be determined over time by monitoring light emitted with a fluorescence microscope & computerized imaging techniques

B. Such work has provided dramatic portraits of the complex spatial & temporal changes in free cytosolic Ca2+ ion concentration that occur in a single cell in response to various stimuli

II. Depending on the type of responding cell, a particular stimulus may:

A. Induce repetitive oscillations in the concentration of free calcium ions

B. Cause a wave of Ca2+ ion release that spreads from one end of the cell to the other or

C. Trigger a localized & transient release of Ca2+ ions in one part of the cell – example Purkinje cell

1. A Purkinje cell is a mammalian cerebellar neuron that maintains contact with thousands of other cells through an elaborate postsynaptic dendrite network

2. Free Ca2+ ions are released in a localized region of the dendritic tree after synaptic activation; the burst of calcium release stays restricted to this region of the cell

III. There are 2 main types of Ca2+ ion channels present in the ER membrane that allow Ca2+ ions into cytoplasm: IP3 receptors (see above) & ryanodine receptors (RyRs)

A. Ryanodine receptors – called ryanodine receptors since they bind the toxic plant alkaloid ryanodine; found primarily in excitable cells & are best studied in skeletal & cardiac muscle cells

1. They mediate the rise in Ca2+ levels after the arrival of an action potential

2. Mutations in the cardiac RyR isoform have been linked to occurrences of sudden death during periods of exercise

B. Depending on the type of cells in which they are found, RyRs can be opened by a variety of agents, including calcium itself

1. Influx of a limited amount of Ca2+ through open cell membrane channels induces opening of ER RyRs, causing Ca2+ release into cytosol, a phenomenon called calcium-induced calcium release (CICR)

C. Extracellular signals that are transmitted by Ca2+ ions typically act by opening a small number of Ca2+ ion channels at the cell surface at the site of the stimulus

1. As Ca2+ ions rush through these channels & enter cytosol, they act on nearby Ca2+ ion channels in the ER, causing these channels to open & release additional Ca2+ ions into adjacent cytosol regions

2. Sometimes elevation of Ca levels remains localized to small cytosol regions; other times, a propagated wave of calcium release spreads through the entire cytoplasmic compartment

D. One of the most dramatic Ca2+ ion waves occurs within the first minute or so after fertilization & is induced by the sperm's contact with the plasma membrane of the egg

1. The sudden rise in cytoplasmic Ca2+ concentration after fertilization triggers a number of events, including activation of cyclin-dependent kinases that drive the zygote toward its first mitotic division

2. Calcium waves are transient because the ions are rapidly pumped out of the cytosol & back into the ER and/or the extracellular space

The Role of Calcium as an Intracellular Messenger: Ca2+-Binding Proteins

I. Calcium can affect a number of different cellular effectors, like Ca-dependent protein kinases, while cAMP's action is invariably mediated by stimulation of a protein kinase

A. Depending on cell type, Ca2+ ions can activate or inhibit various enzyme & transport systems, change membrane ionic permeability, induce membrane fusion, or alter cytoskeletal structure & function

1. Calcium does not bring about these responses by itself, but acts in conjunction with a number of Ca2+-binding proteins, like calmodulin (widely distributed; best-studied)

2. Calmodulin participates in many signaling pathways

B. Calmodulin is found universally in plants, animals & eukaryotic microorganisms; it has virtually the same amino acid sequence throughout the eukaryotes (sequence is highly conserved)

1. Each calmodulin has 4 Ca2+-binding sites, but its binding affinity for Ca2+ ions is not high enough to bind the ion in a nonstimulated cell

2. If, however, Ca2+ ion concentration rises in response to a stimulus, the ions bind to calmodulin, changing the protein's conformation & increasing its affinity for a variety of effectors

3. The Ca2+-calmodulin (Ca2+-CaM) complex may bind to a protein kinase, a cyclic nucleotide phosphodiesterase, ion channels or even to the plasma membrane Ca2+-transport system

4. Rising Ca2+ levels can activate the system responsible for ridding the cell of excess quantities of Ca2+, thus constituting a self-regulatory mechanism for maintaining low intracellular [Ca2+ ion]

C. The Ca2+-CaM complex can also stimulate gene transcription through the activation of various protein kinases (Ca MKs) that phosphorylate transcription factors

1. In the best-studied case, one of these protein kinases phosphorylates CREB on the same serine residue as PKA

II. Regulating calcium concentrations in plant cells – calcium ions acting in conjunction with calmodulin are important intracellular messengers in plant cells

A. The levels of cytosolic Ca2+ change dramatically within certain plant cells in response to a variety of stimuli – changes in light, pressure, gravity & the concentration of plant hormones (abscisic acid)

B. The concentration of Ca2+ in the cytosol of a resting plant cell is kept very low by the action of transport proteins situated in the plasma & vacuolar (tonoplast) membranes

C. Ca2+ role in plant cell signaling – example: guard cells regulating microscopic leaf pore (stomata) diameter

1. Stomata are a major site of water loss in plants & the diameter of their aperture is tightly controlled to prevent desiccation

2. The diameter of the stomatal pore decreases as the fluid (turgor) pressure in the guard cell decreases

3. The drop in turgor pressure is caused, in turn, by a decrease in the ionic concentration (osmolarity) of the guard cell

4. Adverse conditions (high temperatures & low humidity) stimulate release of abscisic acid —> opens Ca2+ channels in guard cell plasma membrane

5. The resulting Ca2+ influx into cytosol triggers release of more Ca2+ ions from intracellular stores

6. Elevated cytosolic [Ca2+ ion] leads to cell membrane K+ influx channel closure & K+ efflux channel opening —> produces net K+ ion outflow (& accompanying Cl- ions) & a drop in turgor pressure

Convergence, Divergence and Crosstalk Among Different Signaling Pathways

I. Cell signaling pathways are often much more complex than a direct linear connection leading from a cell surface receptor to an end target; for example:

A. Signals from a variety of unrelated receptors (each binding to its own ligand) can converge to activate a common effector (like Ras or Raf)

B. Signals from same ligand (EGF, insulin) can diverge to activate a variety of different effectors —> leads to diverse cellular responses

C. Signals can be passed back & forth between different pathways (a phenomenon called crosstalk)

II. Signaling pathways are like nervous system & provide a mechanism for routing information through a cell; cell gets information about its environment via activation of various surface receptors (they detect stimuli)

A. Cell-surface receptors can bind only to specific ligands & are unaffected by a large variety of unrelated molecules

B. A single cell may have dozens of different receptors sending signals to the cell interior simultaneously

C. Once in a cell, signals from receptors can be selectively routed along many different signaling pathways; may cause cell division, shape changes, specific metabolic pathway activation or even cell suicide

D. The cell integrates information arriving from different sources & mounts an appropriate & comprehensive response

III. Example 1: convergent signaling – there are 3 different kinds of receptors (G protein-coupled receptors, RTKs, integrins) that bind to different ligands but…..

A. All of them can lead to the formation of phosphotyrosine docking sites for SH2 domain of the Grb2 adaptor protein

B. Grb2 recruitment results in Ras activation & transmission of signals down the MAP kinase pathway

C. Thus, signals from diverse receptors can lead to the transcription & translation of a similar set of growth-promoting genes in each target cell

IV. Example 2: divergent signaling – a ligand binding to an insulin receptor, a growth factor receptor or an integrin sends signals out along a variety of different pathways

A. Interaction of insulin with its receptor sends signals along separate pathways headed by Ras, PI3K & PLCeach of which initiates distinct cellular responses

V. Example 3: crosstalk between signaling pathways - cell signaling pathways highly interdependent

A. Information circuits operating in cells are more likely to resemble an interconnected web in which components produced in one pathway can participate in events occurring in other pathways

1. The more that is learned about information signaling in cells, the more crosstalk between signaling pathways is discovered – cAMP is an example

B. cAMP leads not only to glucose mobilization, but is also involved in other pathways; it inhibits growth in a variety of cells (fibroblasts, fat cells), by blocking signals transmitted through MAP kinase cascade

1. cAMP activates PKA, the cAMP-dependent kinase, which can phosphorylate & inhibit Raf, the protein that heads the MAP kinase cascade

2. These 2 pathways also intersect at another important signaling effector, the transcription factor CREB (a terminal effector of cAMP-mediated pathways)

3. It was assumed for years that CREB could only be phosphorylated by the cAMP-specific kinase, PKA; it is now known that CREB is a substrate for a much wider range of kinases

C. One kinase that phosphorylates CREB is Rsk-2, which is activated as a result of phosphorylation by MAPK; both PKA & Rsk-2 phosphorylate CREB on precisely the same amino acid residue, Ser133

1. This should endow the transcription factor (CREB) with the same potential in both pathways

VI. How are different stimuli able to evoke distinct responses, even though they use similar pathways?

A. PI3K is an enzyme that is activated by a remarkable variety of stimuli, including cell adhesion to the ECM, insulin & EGF

B. How does PI3K activation in an insulin-stimulated liver cell promote GLUT4 translocation & protein synthesis, while in an adherent epithelial cell, it promotes cell survival?

1. This must be due to differences in the protein composition of different cell types

2. Part of the answer probably is that different cells have different versions (isoforms) of these various proteins, including PI3K

3. Some of these isoforms are encoded by different, but related, genes; others are generated by alternate splicing or other mechanisms

4. Different isoforms of PI3K, PKB or PLC may bind to different sets of upstream & downstream components; which could allow similar pathways to evoke distinct responses

C. But isoform variation alone is not likely to explain the extraordinary diversity of cell responses; other factors will likely be discovered that explain how specificity is achieved with similar signaling molecules

Other Signaling Pathways: The Role of NO as an Intercellular Messenger

I. During 1980s, a new type of messenger was discovered; it was neither an organic compound (cAMP) nor an ion (Ca2+), but an inorganic gas nitric oxide (NO)

A. NO is unusual because it acts both as an extracellular messenger, mediating intercellular communication, as well as a 2nd messenger, acting within the cell in which it is generated

B. NO is formed from the amino acid L-arginine in a reaction that requires oxygen & NADPH; the reaction is catalyzed by the enzyme nitric oxide synthase (NOS)

C. Since its discovery, it has become evident that NO is involved in a myriad of biological processes including anticoagulation, neurotransmission, smooth muscle relaxation & visual perception

1. NO should not be confused with nitrous oxide (N2O) or "laughing gas"

II. The discovery that NO functions as a messenger molecule began with an accidental observation

A. It has been known for many years that acetylcholine (ACH) acts in the body to relax the smooth muscle cells of blood vessels, but the result could not be duplicated in vitro

1. When portions of a major blood vessel (like the aorta) were incubated in physiological concentrations of acetylcholine in vitro, the preparation usually showed little or no response

B. Robert Furchgott (pharmacologist, NY State Medical Center, late 1970s) was studying the in vitro response of rabbit aorta pieces to various agents

1. In earlier studies, Furchgott used strips of aorta dissected from the organ

2. For technical reasons, he switched to aortic rings & discovered that the new preparations responded to acetylcholine by undergoing relaxation

3. It was found that the strips failed to display the relaxation response because the delicate endothelial layer that lines the aorta had been rubbed away during dissection

4. This suggested that the endothelial cells were somehow involved in the response by the adjacent muscle cells

5. Eventually, it was found that acetylcholine binds to receptors on the surface of endothelial cells, leading to the production & release of an agent that diffuses through the cell's plasma membrane

6. This agent causes the muscle cells to relax & it was identified in 1986 as NO by Louis Ignarro (UCLA) & Salvador Moncada (Wellcome Research labs, England)

C. Steps in the acetylcholine-induced relaxation response of blood vessel smooth muscle

1. ACH binds to outer surface of endothelial cell —> signals a rise in cytosolic Ca2+ concentration

2. Rise in cytoplasmic Ca2+ concentration activates nitric oxide synthase

3. NO made by NOS in endothelial cell diffuses across endothelial cell membrane into adjacent smooth muscle cells

4. NO binds to guanylyl cyclase in smooth muscle cells & stimulates the enzyme to make cyclic GMP (cGMP is an important 2nd messenger similar in structure to cAMP)

5. cGMP binds to a cGMP-dependent protein kinase (a PKG), which phosphorylates specific substrates causing relaxation of the muscle cell & dilation of the blood vessel

III. NO as an activator of guanylyl cyclase – the finding that NO activates guanylyl cyclase was made in late 1970s by Ferid Murad et al. (Univ. of Va.)

A. They were working with azide (N3), a potent inhibitor of electron transport, & chanced to discover that it stimulated cGMP production in cellular extracts

1. Later showed that N3 was converted enzymatically to NO, the actual guanylyl cyclase activator

2. Explained nitroglycerine action; it had been used since 1860s to treat angina pain caused by inadequate blood flow to heart - nitroglycerine is metabolized to NO

3. NO stimulates relaxation of smooth muscles lining heart blood vessels, increasing flow to heart

B. Nitroglycerine's therapeutic benefits were discovered through an interesting observation

1. Persons with heart disease who worked with the compound in Alfred Nobel's dynamite factory were found to suffer more from the pain of angina on days they weren't at work

2. It is fitting that the Nobel Prize, which is funded by a donation from Alfred Nobel's estate, was awarded in 1998 for the discovery of NO as a signaling agent

IV. Inhibiting phosphodiesterase – discovery of NO as a 2nd messenger has impacted millions of lives since it led to development of Viagra (sildenafil) – during sexual arousal, nerve endings in penis release NO

A. NO release —> smooth muscle cell relaxation in penile blood vessel lining —> penile engorgement

1. NO mediates the smooth muscle cell response by activating guanylyl cyclase & cGMP production

B. Viagra (& related drugs) does not influence NO release or guanylyl cyclase activation, but instead acts as an inhibitor of cGMP phosphodiesterase, the enzyme that destroys cGMP

1. Inhibition of cGMP phosphodiesterase leads to maintained, elevated cGMP levels

2. High cGMP promotes the development & maintenance of an erection

C. Viagra is quite specific for one particular cGMP phosphodiesterase isoform, PDE5, which is the version that acts in the penis

1. Another isoform of the enzyme, PDE3, plays a key role in the regulation of heart muscle contraction, but fortunately is not inhibited by Viagra

D. Viagra was discovered when a potential angina medication had unexpected side effects

V. Recent investigations have shown that that NO has a variety of actions within the body that do not involve cGMP production

A. NO is added to the —SH group of certain cysteine residues in a number of proteins, including hemoglobin, Ras, ryanodine channels & caspases

B. This posttranslational modification, S-nitrosylation, alters the protein's activity, turnover or interactions

Other Signaling Pathways: Apoptosis (Programmed Cell Death)

I. Apoptosis (programmed cell death) – a normal process in which an orchestrated sequence of events leads to cell death; death by apoptosis (pronounced a-poe-toe-sis) is a neat, orderly process characterized by the:

A. Overall shrinkage in volume of the cell & its nucleus

B. Loss of adhesion to neighboring cells

C. Formation of blebs at the cell surface

D. Dissection of the chromatin into small fragments and

E. Rapid engulfment of the "corpse" by phagocytosis

II. Why do our bodies have unwanted cells & where do we find cells that get targeted for elimination? – almost anywhere you look; it has been estimated that 1010 – 1011 cells in the human body die daily by apoptosis

A. During embryonic development, many more neurons grow out of central nervous system (CNS) toward target organ in the body periphery to innervate it than are needed to innervate the organ normally

1. The neurons that reach their destination receive a signal from target tissue allowing them to survive

2. Those neurons that fail to find their way to the target tissue do not receive the survival signal & are ultimately eliminated by apoptosis

B. T lymphocytes are immune system cells that recognize & kill abnormal or pathogen-infected target cells; these target cells are recognized by specific receptors that are present on T lymphocyte surface

1. During embryonic development, T lymphocytes are produced that possess receptors capable of binding tightly with proteins present on the surface of normal cells within the body

2. T lymphocytes that have this dangerous capability are eliminated by apoptosis

C. Apoptosis is involved in the elimination of cells that have sustained irreparable genomic damage

1. Important because damage to genetic blueprint can result in unregulated cell division & development of cancer

D. Apoptosis appears to be involved in neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease)

1. Elimination of essential neurons during disease progression gives rise to loss of memory or decrease in motor coordination

E. The above examples show that apoptosis is important in maintaining homeostasis in multicellular organisms & that failure to regulate apoptosis can result in serious damage to the organism

III. Term apoptosis coined in 1972 by John Kerr, Andrew Wyllie & A. R. Currie (Univ. of Aberdeen, Scotland) in landmark paper that first described coordinated events occurring during programmed death of many cells

A. Insight into molecular basis of apoptosis was first revealed in studies of nematode worm Caenorhabditis elegans, whose cells can be followed with absolute precision during embryonic development

1. Of 1090 cells produced during worm development, 131 are normally destined to die by apoptosis

B. Robert Horvitz, et al. (MIT, 1986) – discovered that worms carrying a mutation in the CED-3 gene proceed through development without losing any of their cells to apoptosis

1. Suggested that the product of the CED-3 gene played a crucial role in apoptosis in this organism

2. Searched for homologous gene in other organisms, like humans & other mammals; a homologous family of proteins, which is now called the caspases, was found in mammals

IV. Caspases are a distinctive group of cysteine proteases (proteases with a key cysteine residue in their catalytic site) that are activated at an early stage of apoptosis

A. Caspases are responsible for triggering most, if not all, of the changes observed during cell death & they accomplish this feat by cleaving a select group of essential proteins

B. Among caspase targets are:

1. >12 protein kinases, including focal adhesion kinase (FAK), PKB, PKC & Raf1 – FAK inactivation is presumed to disrupt cell adhesion, leading to detachment of an apoptotic cell from its neighbors

2. Lamins – make up the inner lining of the nuclear envelope; cleavage of lamins leads to disassembly of the nuclear lamina & shrinkage of the nucleus

3. Proteins of the cytoskeleton – intermediate filaments, actin, tubulin, gelsolin; cleavage & consequent inactivation of these proteins lead to changes in cell shape

4. An endonuclease (caspase-activated DNase; CAD); activated by caspase cleavage of an inhibitory protein; once activated, it moves from cytoplasm to nucleus; it attacks & severs DNA into fragments

V. New studies focus on events leading to cell's suicide program activation - both internal (DNA abnormalities) & external stimuli (certain cytokines [proteins secreted by immune system cells]) can trigger it

A. Prostate epithelial cells become apoptotic when deprived of the male sex hormone testosterone; that is why drugs interfering with testosterone production are used to treat metastasized prostate cancer

B. Studies suggest that external stimuli activate apoptosis by a signaling pathway (the extrinsic pathway) that is distinct from that utilized by internal stimuli (the intrinsic pathway)

C. Intrinsic & extrinsic pathways discussed separately; however, it should be noted that there is cross-talk between these pathways & that extracellular apoptotic signals can cause intrinsic pathway activation

VI. The extrinsic pathway of apoptosis – an extracellular messenger protein called tumor necrosis factor (TNF), which is named for its ability to kill tumor cells & is an apoptotic stimulus; best understood

A. TNF is produced by certain immune system cells in response to adverse conditions (exposure to ionizing radiation, elevated temperature, viral infection, toxic chemical agents [cancer chemotherapy])

B. TNF evokes response by binding to transmembrane receptor TNFR1 (member of a family of related death receptors that turn on the apoptotic process)

C. Evidence suggests that TNFR1 is present in plasma membrane as a preassembled trimer

1. Each TNFR1 receptor subunit has a cytoplasmic domain with a segment of ~70 amino acids (the death domain) that mediates protein-protein interactions

2. TNF binding to trimeric receptor produces a conformational change in receptor's death domain that initiates recruitment of a number of proteins

3. The last proteins to join complex assembling at inner plasma membrane surface are 2 procaspase-8 molecules; called procaspases since each is a precursor of a caspase; they are proenzymes

4. Procaspases contain an extra portion that must be removed by proteolytic processing to activate the enzyme; synthesis of caspases as proenzymes protects cell from accidental proteolytic damage

C. Unlike most proenzymes, procaspases have a low proteolytic activity level

1. According to one model, when ≥2 of them are held close together, they can cleave one another's polypeptide chain —> converts other one to fully active caspase

2. The final mature enzyme (caspase-8) has 4 polypeptide chains, derived from 2 procaspase precursors

D. Caspase-8 activation is similar in principle to activation of effectors by hormones or growth factors

1. Extracellular ligand binding causes receptor conformation change —> binding & activation of proteins situated downstream in pathway

2. Caspase-8 is an initiator caspase since it initiates apoptosis by cleaving & activating downstream or executioner caspases that carry out the controlled self-destruction of cell

VII. The intrinsic pathway of apoptosis

A. What internal stimuli trigger apoptosis by the intrinsic pathway?

1. Irreparable genetic damage

2. Lack of oxygen (hypoxia)

3. Extremely high cytosolic Ca2+ concentrations

4. Severe oxidative stress (production of lots of destructive free radicals)

B. Activation of the intrinsic pathway is regulated by members of the Bcl-2 family of proteins

1. Bcl-2 family members are subdivided into 2 groups: pro-apoptotic members that promote apoptosis (Bad, Bax) & anti-apoptotic members that protect cells from apoptosis (Bcl-xL, Bcl-w, Bcl-2)

2. Bcl-2 itself was originally identified in 1985 as a cancer-causing oncogene in human lymphomas; it acts as an oncogene by promoting survival of potential cancer cells that would otherwise die

C. Stressful stimuli like those above activate certain pro-apoptotic members of the Bcl-2 family, like Bax, which translocates from the cytosol to the outer mitochondrial membrane

1. The insertion of Bax into the outer mitochondrial membrane increases the permeability of that membrane & promotes the release of certain mitochondrial proteins, most notably cytochrome c

2. Cytochrome c, while loosely associated with the outer surface of inner mitochondrial membrane, actually resides in the intermembrane space

3. Evidence suggests that Bax (and/or Bak) forms a protein-lined channel within the mitochondrial membrane

4. Mitochondrial membrane permeability may be accelerated by a rise in cytosolic Ca2+ levels following the release of the ion from the ER

5. Nearly all of the cytochrome c present in all of the cell's mitochondria can be released from apoptotic cell in a period as short as 5 minutes

6. Anti-apoptotic proteins like Bcl-2 are thought to either directly or indirectly inhibit the release of cytochrome c & other proapoptotic mitochondrial proteins that lead to cell death

D. Release of proapoptotic mitochondrial proteins may be the crucial event that commits cell to apoptosis

1. Once in cytosol, cytochrome c forms part of a multiprotein complex (the apoptosome) that also includes several procaspase-9 molecules

2. Studies suggest that procaspase-9 is activated by simply joining the multiprotein complex & does not require proteolytic cleavage

3. Caspase-9, like caspase-8, is an initiator caspase that activates downstream executioner caspases, which bring about apoptosis

4. The extrinsic (receptor-mediated) & intrinsic (mitochondria-mediated) pathways ultimately converge by activating the same executioner caspases, which cleave the same cellular targets

5. Other intrinsic pathways that are independent of Apaf-1 & caspase-9, & possibly independent of cytochrome c, have also been described

E. Why is cytochrome c, a component of the electron transport chain, & the mitochondrion, an organelle that functions as the cell's power plant, involved in initiating apoptosis?

1. Right now there is no obvious answer; mitochondrial involvement in apoptosis is also perplexing since they evolved from prokaryotic endosymbionts & prokaryotes do not undergo apoptosis

VIII. As cells execute the apoptotic program, they lose contact with their neighbors & start to shrink; finally, cell disintegrates into condensed, membrane-enclosed apoptotic body; whole program can happen in <1 hour

A. Apoptotic bodies are recognized by the presence on their surface of phosphatidylserine, which is a phospholipid that is normally present only on the inner leaflet of the plasma membrane

B. During apoptosis, a phospholipid "scramblase" moves phosphatidylserine molecules to the plasma membrane outer leaflet where they are recognized as an "eat me" signal by specialized macrophages

1. Apoptotic cell death thus occurs without spilling cellular content into the extracellular environment

2. This is important since the release of cellular debris can trigger inflammation, which can cause a significant amount of tissue damage

IX. Just as there are signals committing cell to self-destruction, there are opposing signals maintaining cell survival

A. Interaction of TNF with a TNF receptor often transmits 2 distinct & opposing signals into the cell interior: one stimulating apoptosis & another stimulating cell survival

B. Thus, most cells possessing TNF receptor do not undergo apoptosis when treated with TNF

1. This was disappointing since it was hoped that TNF could be used as an agent to kill tumor cells

C. Cell survival is typically mediated through activation of a key transcription factor called NF-B, which activates the expression of genes encoding cell-survival proteins

1. It appears that the fate of a cell (survival or death) depends on a delicate balance between pro-apoptotic & anti-apoptotic signals

The Human Perspective: Disorders Associated with G-Protein Coupled Receptors

I. The human genome may encode as many as 2000 different GPCRs

A. They are very important, since >1/3 of all prescription drugs act as ligands that bind to this huge superfamily of receptors

B. A number of inherited disorders have been traced to defects in both GPCRs & heterotrimeric G proteins

II. Retinitis pigmentosa (RP) is an inherited disease characterized by progressive degeneration of retina & eventual blindness; it can be caused by mutations in gene encoding rhodopsin, the visual pigment of rods

A. Many of these mutations lead to premature termination or improper folding of the rhodopsin protein & its elimination from the cell before it reaches the plasma membrane

B. Other mutations may lead to the synthesis of a rhodopsin molecule that cannot activate its G protein & thus cannot pass the signal downstream to the effector

C. RP results from a mutation leading to loss of function of the encoded receptor

III. In contrast to the case with RP, many mutations that alter the structure of signaling proteins can have the opposite effect, leading to a gain of function – example: thyroid adenomas

A. Mutations have been found to cause a type of benign thyroid tumor, an adenoma

1. Normal thyroid cells secrete thyroid hormone only in response to stimulation by the pituitary hormone TSH

2. The cells of these thyroid adenomas secrete large quantities of thyroid hormone without having to be stimulated by TSH; the receptor is said to act constitutively

3. The TSH receptor in these cells contains an amino acid substitution that affects the structure of the third intracellular loop of the protein

4. Thus, the TSH receptor constitutively activates a G protein on the inner membrane surface, sending a continual signal through its pathway

5. The signal leads not only to excessive thyroid hormone secretion, but also to the excessive cell proliferation that causes the tumor

6. Verified - introduce mutant gene into cultured cells normally lacking receptor —> mutant protein made & incorporated into membrane; continuous cAMP production in genetically engineered cells

B. The mutation causing thyroid adenomas is not found in the normal portion of the patient's thyroid, but only in tumor tissue, indicating that the mutation was not inherited but arose in one of the thyroid cells

1. The cell then proliferated to give rise to the tumor

2. A mutation in a cell of the body, like a thyroid cell, is called a somatic mutation to distinguish it from an inherited mutation that would be present in all of an individual's cells

3. Somatic mutations are a primary cause of human cancers

IV. At least 1 cancer-causing virus has been shown to encode a protein that acts as a constitutively active GPCR

A. The virus is a type of herpes virus that is responsible for Kaposi's sarcoma, which causes purplish skin lesions & is prevalent in AIDS patients

B. The virus genome encodes a constitutively active receptor for interleukin-8, which stimulates signaling pathways that control cell proliferation

V. Mutations in genes coding for heterotrimeric G protein subunits can lead to inherited disorders; ex: 2 male patients with rare endocrine disorder combination: precocious puberty & hypoparathyroidism

A. Both patients had a single amino acid substitution in one of the G isoforms; the alteration in amino acid sequence had 2 effects on the mutant G protein:

1. At temperatures below normal body temperature, the mutant G protein remained in the active state, even in the absence of a bound ligand

2. In contrast, at normal body temperatures, the mutant G protein was inactive, both in the presence & absence of a bound ligand

B. Testes, housed outside body core, have lower temperature than body's visceral organs (33°C vs. 37°C); normally, testes endocrine cells make testosterone at puberty (response to newly made pituitary LH)

1. Circulating LH binds to LH receptors on testicular cell surfaces, inducing cAMP synthesis & subsequent production of the male sex hormone

2. Testicular cells of the patients bearing the G protein mutation were stimulated to synthesize cAMP in the absence of the LH ligand, leading to premature testosterone synthesis & precocious puberty

C. In contrast, the mutation in this same G subunit in the cells of parathyroid glands, which function at 37°C, caused the G protein to remain inactive

1. Thus, the cells of the parathyroid gland could not respond to stimuli that would normally cause them to secrete parathyroid hormone, leading to the condition of hypoparathyroidism

D. The fact that most of the bodily organs functioned in a normal manner in these patients suggests that this particular G isoform is not essential in the activity of most other cells

VI. Mutations are thought of as rare & disabling changes in the nucleotide sequence of a gene; in contrast genetic polymorphisms, are thought of as common, normal variations within the population

A. It has become clear that genetic polymorphism may have considerable impact on human disease

1. They cause certain individuals to be more susceptible to particular disorders than other individuals

B. This has been well documented in the case of GPCRs - examples

1. Certain alleles of the gene encoding the 2 adrenergic receptor have been associated with an increased likelihood of developing asthma or high blood pressure

2. Certain alleles of a dopamine receptor are associated with increased risk of substance abuse or schizophrenia

3. Certain alleles of a gene (CCR5) are associated with prolonged survival in HIV-infected individuals

C. Identifying associations between disease susceptibility & genetic polymorphisms is a current focus of clinical research