G-protein Coupled Receptors and their Signaling Mechanism

G-protein coupled receptors are integral plasma membrane proteins that transduce signals from extra cellular ligands to signals in intracellular heterotrimeric GTP binding proteins (G proteins). By coupling to many downstream effectors, the G proteins initiate pleiotropic changes in many targets. Thus, the extra cellular signal is typically amplified to produce robust, varied, and cell-specific responses. G-protein coupled receptors are quite distinct from growth-factor receptors, which signal through tyrosine kinases, do not use heterotrimeric G proteins, and typically regulate gene expression. However, G-protein coupled receptors and growth-factor receptors do share several common final pathways of signaling. For most of the extracellular stimuli there are multiple different receptors, and the same extracellular signal can give rise to different intracellular responses depending on the receptor subtypes expressed. For example there are nine subtypes (genes) of receptors for adrenaline/noradrenaline. Three of them couple to the G-protein Gq, often making calcium signals (1 adrenergic receptors), three of them couple to Gi, often inhibiting adenylyl cyclase (2 adrenergic receptors), and three of them couple to Gs, often stimulating adenylyl cyclase ( adrenergic receptors). Since the adrenergic receptors are more closely related to each other than to other GPCRs, one can see that a prototype adrenergic receptor evolved early that through further gene duplications subsequently diversified its repertoire of G-protein coupling

Structure:
All GPCRs have a membrane topology with seven -helical transmembrane segments, Nterminus outside and C-terminus inside. X-ray crystal structures, available so far for only a few GPCRs, show that the transmembrane segments are broken helices that cross the membrane at various angles. The binding site for small agonists often lies nestled between helices and part way across the membrane, and that for protein ligands is on an elongated extracellular N-terminus. However in class-C receptors such as mGluR and GABABR, the small ligand binds within an extracellular "clamshell" formed by the exceedingly long Nterminus.Crystal structure of the 1-adrenergic receptor. Transmembrane helical segments are numbered 1-7, where 1 should connect to the extracellular N-terminus; however in this structure the N-terminus was truncated and is absent. An antagonist, cyanopindolol is shown in the agonist binding site (purple stick structure). A bold arrow points to the intracellular crevice where part of the G subunit can inset into the receptor when agonist is bound from the outside. C is the C-terminus. Biochemical experiments show that heterotrimeric G-proteins interact with the second and third intracellular loops (between helices 3 and 4 and between helices 5 and 6) and with the cytoplasmic C-terminus of receptors . These interactions determine which G-proteins each receptor will couple to, and they transmit the message from the activated receptor to the G-protein that initiates nucleotide exchange.Since 1998, there is growing evidence that many GPCRs can form dimers, two receptors in one complex. Notable examples are mgluR5 or GABAB receptors. Both homodimers and heterodimers are formed. The structure of such complexes is not known, but it is certain that some receptor dimers are active in signaling and sometimes

even are obligatory for signaling (GABABR). The consequences, generality, and significance of receptor dimerization need further investigation. One concept is that the larger intracellular surface area of a receptor dimer would offer a better interaction interface for a single G protein since G proteins are so much larger than receptors. Potentially, dimerization can alter agonist and antagonist specificity, G-protein coupling, and membrane trafficking and recycling, giving dimeric receptors properties that the monomeric forms did not have.

Figure: Structure of G-protein coupled receptors

Signaling from G-protein Coupled Receptors:

G-protein coupled receptors subunits .At rest these three subunits are assembled into a complex, G. Since G and G are inseparable once coassembled, it is customary to talk

about a resting GG complex. Typically, both G and G are held at the inner leaflet of the plasma membrane by hydrophobic lipid modifications. Unlike the schematic drawing in, the heterotrimeric G protein is physically larger than the GPCR (typical molecular weights: G ~90K, GPCRs, ~50K). The heterotrimeric G proteins should be distinguished from the small, low molecular weight, monomeric G proteins of the Ras, Rho, etc. families. The monomeric G proteins have the GTP-binding and GTPase properties of G subunit of heterotrimeric G proteins but they do not couple to or G, and they signal to different downstream effectors. G subunits are flexible signaling proteins. In the inactive resting complex, the G subunit is bound to the guanine nucleotide, guanosine diphosphate (GDP), but when a receptor is activated, the receptor can catalyze nucleotide exchange reactions on the G subunit. GDP leaves and guanosine triphosphate GTP enters instead. GTP binding activates the G protein (hence the name). Current research seeks to understand the molecular interactions between receptor and G protein and how the conformational energy of ligand binding results in GDP-GTP exchange. In classical teaching, the G-GTP-G complex is unstable so that the active G-GTP and G separate from one another and from the receptor as well, but they usually remain attached to the plasma membrane by their lipid anchors. However, in some examples it is believed that the quaternary and active G-protein-GTP complex remains undissociated although conformationally changed, and in some other examples the G dimer may leave the membrane and go into the cytoplasm or to other membranes. GGTP and G each signal to downstream effectors. Because the activated G proteins are generally membrane associated, the next step usually is an interaction with membrane-

associated effector proteins or recruitment of cytoplasmic effector proteins to the membrane. We now consider signaling from G-GTP and G separately.

Fig: Downstream coupling of G-GTP

Five Canonical Signaling Pathways:

The downstream effects of stimulating a GPCR depend on which G protein type(s) it couples to. Heterotrimeric G proteins are named by the type of subunit they contain, and there are nearly 20 genes encoding G subunits. However, for understanding much of their signaling it suffices to consider five broad G families and five signaling pathways that they most typically activate: 1. Gs , Golf : activates plasma membrane adenylyl cyclases, increasing cellular cyclic AMP (cAMP), which e.g. stimulates phosphorylation of target proteins by cAMPdependent protein kinase. Gs and its downstream signaling can be covalently activated by cholera toxin. 2. Gi, Go : inhibit most adenylyl cyclases, decreasing cellular cAMP. Gi and Go can be covalently inactivated and their signaling turned off by Pertussis toxin. Go is said to constitute 1% of brain proteins. 3. Gq, G11: activate phospholipase C (PLC), which cleaves certain phosphoinositide lipids (PIP2) of the plasma membrane, and generates several second messengers that e.g. release Ca2+ from intracellular stores and activate phosphorylation by protein kinase C. 4. G12, G13: enhance Rho kinase and change expression of some genes and the phosphorylation of myosin 5. Gtransducin, Ggustducin: activates cyclic GMP (cGMP) phosphodiesterase (transducin) that cleaves and depletes cytoplasmic cGMP (retina only) or cAMP phosphodiesterase (gustducin) that cleaves and depletes cAMP (taste receptors) Each of these pathways involves second messengers and effector enzymes. The long cascade of signaling may take up to tens of seconds to be completed. However in a few

cases, such as vision using rhodopsin and transducin, a high density, very tight compartmentalization, and miniaturization of the geometry have allowed responses that take only tens of milliseconds.

Downstream coupling of G:
The G subunits also are potent signals. They bind to several effectors. They activate Gprotein coupled inwardly rectifying K+ (GIRK) channels. They inhibit opening of voltagegated Ca2+ channels of the Cav family. They bind to the SNARE complex of the exocytotic machinery in synapses and reduce exocytosis of neurotransmitters. In neurobiology, the latter two signaling actions provide a major component of presynaptic inhibition by reducing Ca2+ entry and by blocking exocytosis of transmitter. In addition G dimers act directly on at least two more downstream effectors, stimulating PLC and phosphoinositide 3-kinase (PI3K). Although there are numerous G and G genes, to a first approximation, the G populations associated with all G subunits are similar. By mass action, the strength of G signaling to effectors is probably greatest when launched by the most abundant G subunits (e.g. Go in neurons) or when derived from types of G subunits that dissociate most readily from their G partners.

Termination of GPCR Signaling:

Termination of signaling requires turning off activated receptors, turning off activated Gproteins, and return of second messenger levels, protein phosphorylation levels, and other changed metabolites to their original values. Consider the first two. Receptors quickly

deactivate upon removal and unbinding of agonist ligand. They are also inactivated by other processes even while ligand is still present, mechanisms that prevent overstimulation. In one canonical shutdown pathway, activated receptors are recognized and phosphorylated by G-protein coupled receptor kinases (GRKs). Phosphorylated receptors may be intrinsically less active, and they can be turned off fully by binding of at the plasma membrane. The arrestin-receptor complex may be unable to couple to downstream effectors, and it may be endocytosed (clathrin-mediated), removing the receptor entirely from the cell surface, a true down regulation of receptor protein. Signaling by activated G-GTP is terminated by GTP hydrolysis, a reaction catalyzed by the G subunit itself that yields the inactive form, G-GDP. Thus activated G-proteins have an intrinsic self-timer function that terminates their activity. G-GDP in turn is a scavenger that binds any free G dimers, re-forming the inactive heterotrimeric G protein GGDP. The speed of GTP hydrolysis can be accelerated by proteins that act as GTPase acceleratory proteins (GAPs). Sometimes the effector proteins are GAPS so that activated G-proteins become inactivated more rapidly once they make productive interactions with effectors. In addition, the GTPase activity is speeded by another class of cytoplasmic proteins called regulators of G-protein signaling (RGS proteins) that act as GAPS and sometimes antagonize activation of downstream effectors Signaling without G proteins

Signaling without G proteins:

GPCR activation can evoke signal pathways that do not require coupling to G proteins. Much as activated receptor conformations are recognized by their complementary G proteins (the classical route), so too they can be recognized by other signaling proteins

including the GRKs, arrestins, JAK, Src family kinases, and PDZ-domain containing proteins .Such coupling can evoke clathrin-mediated internalization, activation of MAP (mitogen-activated protein) kinase, or stimulation of Na+/H+ exchange.

Specificity of GPCR Signaling:

Most GPCRs couple primarily to G subunits of only one of the five signaling families listed earlier. Some receptors, termed promiscuous, couple to several. In either case, there would seem to be a large loss of specificity if 1000 types of receptors can couple only to 5 signaling pathways. However, specificity is achieved in several other ways. 1. Each cell expresses only a subset of the available receptors (perhaps 20 or 30 kinds of receptors), meaning that each agonist speaks to specific appropriate cells and not to others. In this way, light stimulates photoreceptors and GnRH stimulates pituitary gonad tropes. 2. Each cell expresses a specific subset of downstream protein targets that are responsive to the second messengers that GPCRs regulate. Therefore, with the same second messenger each cell has a different response. In response to cAMP, Leydig cells of the testis make testosterone, horizontal cells of the retina decrease their electrical coupling, and cardiac pacemaker cells speed the cardiac beat rate. 3. GPCRs may be localized to certain parts of the plasma membrane, although less strongly than for many other membrane proteins. In that way it would be possible in principle for dendrites, cell bodies, axons, and nerve terminals to give different responses to the same set of GPCR agonists. Evidence for strong localization is mostly lacking except in special cases such as the localization of rhodopsin to discs of vertebrate photoreceptors.

4. Finally significant convergence can be desirable. It allows diverse cells and organs to call for a physiological response that they may need. Thus an increase in heart rate and blood circulation can be initiated by noradrenaline from sympathetic nerves for flight-orfight, by glucagon from the pancreas to aid in energy distribution, or by histamine from mast cells to accompany allergic reactions. As many GPCR agonists are released at nerve terminals and varicosities, one might suppose that the corresponding receptors would be highly localized to immediately opposite postsynaptic membranes of a "target" cell. This concept generalizes from the organization of typical fast chemical synapses where presynaptic ACh, glutamate, GABA, or glycine release talks to postsynaptic receptors within nanometers of the release site and opens an ion channel in one postsynaptic neuron within a fraction of a millisecond. Such agonist action stops in a few milliseconds because agonist is quickly removed from the synaptic cleft. However, GPCR signaling is fundamentally different because GPCR agonists typically have an extracellular lifetime of 200 ms to several minutes. In this time, the agonist spreads by diffusion and acts on many cells. The signal necessarily acts in a volume rather than conveying a point-to-point message. Correspondingly, some GPCRs are found more diffusely over the cell surface with less special reference to "postsynaptic" sites. The concept that neurotransmitters may spread beyond a single synapse is called spillover and volume transmission. This is probably the normal mode of action of peptide and monoamine neurotransmitters. They affect the mode of operation of circuits (even mental state) rather than providing specific information to one neuron.

Thus, although it is usual to call nerve-released agonists neurotransmitters whether they act on fast chemical synapses or GPCRs, we summarize below how the actions on these two classes of receptors are quite different and should not be confused: 1. GPCR actions take 100 ms to minutes. Fast chemical synapses signal in a fraction of a millisecond. 2. The GPCRs always evoke complex pleiotropic responses typically involving G proteins, second messengers, and numerous intracellular targets. Fast chemical synaptic receptors only change the membrane potential and sometimes admit calcium ions into the cell. 3. The GPCR coupled monoamines and peptides have longer lifetime and cannot be targeted for point-to-point wiring to a single postsynaptic cell in a circuit. They work on larger groups of cells.

Gs alpha subunit:
The Gs alpha subunit (or Gs protein) is a heterotrimeric G protein subunit that activates the cAMP-dependent pathway by activating adenylate cyclase. The general function of Gs is to activate adenylate cyclase, which, in turn, produces cAMP, which, in turn activates cAMPdependent protein kinase. Further effects of Gs are thus found in function of cAMPdependent protein kinase.It provides a step in signal transduction. Amplification of the signal occurs for instance because the receptor activates several Gs. However, each Gs activates only one adenylate cyclase. G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that respond to a variety of extra cellular stimuli. Each GPCR binds to and is activated by a specific ligand stimulus that ranges in size from small molecule catecholamines, lipids, or

neurotransmitters to large protein hormones. When a GPCR is activated by its extracellular ligand, a conformational change is induced in the receptor that is transmitted to an attached intracellular heterotrimeric G protein complex. The Gs alpha subunit of the stimulated G protein complex exchanges GDP for GTP and is released from the complex. In a cAMP-dependent pathway, the activated Gs alpha subunit binds to and activates an enzyme called adenylyl cyclase, which, in turn, catalyzes the conversion of ATP into cyclic adenosine monophosphate (cAMP). Increases in concentration of the second messenger cAMP may lead to the activation of

* cyclic nucleotide-gated ion channels, * exchange proteins activated by cAMP (EPAC) such as RAPGEF3, or * An enzyme called protein kinase A (PKA). The PKA enzyme is also known as cAMP-dependent enzyme because it gets activated only if cAMP is present. Once PKA is activated, it phosphorylates a number of other proteins including: * enzymes that convert glycogen into glucose * Enzymes that promote muscle contraction in the heart leading to an increase in heart rate. * Transcription factors, which regulate gene expression Specificity of signaling between a GPCR and its ultimate molecular target through a cAMP-dependent pathway may be achieved through formation of a multiprotein complex that includes the GPCR, adenylyl cyclase, and the effector protein.

Activation:
Activated GPCRs cause a conformational change in the attached G protein complex, which results in the Gs alpha subunit's exchanging GDP for GTP and separation from the beta and gamma subunits. The Gs alpha subunit, in turn, activates adenylyl cyclase, which quickly converts ATP into cAMP. This leads to the activation of the cAMP-dependent pathway. This pathway can also be activated downstream by directly activating adenylyl cyclase or PKA.

Molecules that activate cAMP pathway include: * cholera toxin - increase cAMP levels * forskolin - a diterpene natural product that activates adenylyl cyclase * caffeine and theophylline inhibit cAMP phosphodiesterase, which leads to an activation of G proteins that result in the activation of the cAMP pathway * bucladesine (dibutyryl cAMP, db cAMP) - also a phosphodiesterase inhibitor * pertussis toxin, which increase cAMP levels by inhibiting Gi to its GDP (inactive) form. This leads to an increase in adenylyl cyclase, therefore increasing cAMP levels, which can lead to an increase in insulin and therefore hypoglycemia

Deactivation
The Gs alpha subunit slowly catalyzes the hydrolysis of GTP to GDP, which in turn deactivates the Gs protein, shutting off the cAMP pathway. The pathway may also be

deactivated downstream by directly inhibiting adenylyl cyclase or dephosphorylating the proteins phosphorylated by PKA.

Molecules that inhibit the cAMP pathway include: cAMP phosphodiesterase dephospohorylates cAMP into AMP, reducing the cAMP levels.

Inhibitory G protein or Gi:

Some hormones act by inhibiting adenylyl cyclase, lowering cAMP levels, and suppressing protein phosphorylation. For example, the binding of somatostatin to its receptor leads to activation of an inhibitory G protein, or Gi, structurally homologous to Gs, that inhibits adenylyl cyclase and lowers [cAMP]. Somatostatin therefore counterbalances the effects of glucagon. In adipose tissue, prostaglandin E1 inhibits adenylyl cyclase, thus lowering [cAMP] and slowing the mobilization of lipid reserves triggered by epinephrine and glucagon. In certain other tissues PGE1 stimulates cAMP synthesis, because its receptors are coupled to adenylyl cyclase through a stimulatory G protein, Gs. In tissues with 2adrenergic receptors, epinephrine lowers [cAMP], because the 2 receptors are coupled to adenylyl cyclase through an inhibitory G protein, Gi. In short, an extracellular signal such as epinephrine or PGE1 can have quite different effects on different tissues or cell types, depending on three factors: the type of receptor in each tissue, the type of G protein (Gs or Gi) with which the receptor is coupled, and the set of PKA target enzymes in the cells. A fourth factor that explains how so many signals can be mediated by a single second

messenger (cAMP) is the confinement of the signaling process to a specific region of the cell by scaffold proteins.

Gi mainly inhibits the cAMP dependent pathway by inhibiting adenylate cyclase activity, decreasing the production of cAMP from ATP, which, in turn, results in decreased activity of cAMP-dependent protein kinase. Thus, the ultimate effect of Gi is, thus, the opposite of cAMP-dependent protein kinase, which can be found at function of cAMP-dependent protein kinase. It is also attributed a minor role in activation of the phospholipase C pathway.

Fig: The effect of Ri and Gi in cAMP signal pathway

Activation:
Each PKA is a holoenzyme that consists of two regulatory and two catalytic subunits. Under low levels of cAMP, the holoenzyme remains intact and is catalytically inactive. When the concentration of cAMP rises (e.g., activation of adenylate cyclases by G proteincoupled receptors coupled to Gs, inhibition of phosphodiesterases that degrade cAMP),

cAMP binds to the two binding sites on the regulatory subunits, which leads to the release of the catalytic subunits.

Figure: Activation and inactivation mechanisms of PKA

Catalysis:
The free catalytic subunits can then catalyse the transfer of ATP terminal phosphates to protein substrates at serine, or threonine residues. This phosphorylation usually results in a change in activity of the substrate. Since PKAs are present in a variety of cells and act on different substrates, PKA and cAMP regulation are involved in many different pathways. The mechanisms of further effects may be divided into direct protein phosphorylation and protein synthesis:

In direct protein phosphorylation, PKA directly either increases or decreases the activity of a protein.

In protein synthesis, PKA first directly activates CREB, which binds the cAMP response element, altering the transcription and therefore the synthesis of the protein. In general, this mechanism takes longer time (hours to days).

Inactivation:
Thus, PKA is controlled by cAMP. Also, the catalytic subunit itself can be down-regulated by phosphorylation. Downregulation of protein kinase A occurs by a feedback mechanism: One of the substrates that are activated by the kinase is a phosphodiesterase, which quickly converts cAMP to AMP, thus reducing the amount of cAMP that can activate protein kinase A.

Gq alpha subunit:
A second class of serpentine receptors are coupled through a G protein to a plasma membrane phospholipase C (PLC) that is specific for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. This hormone-sensitive enzyme catalyzes the formation of two potent second messengers: diacylglycerol and inositol 1,4,5-trisphosphate, or IP3. When a hormone binds its specific receptor in the plasma membrane, the receptor-hormone complex catalyzes GTP-GDP exchange on an associated G protein, Gq (step 2), activating it exactly as the adrenergic receptor activates Gs. The activated Gq in turn activates a

specific membrane-bound PLC (step 3 ), which catalyzes the production of the two second messengers diacylglycerol and IP3 by hydrolysis of phosphatidylinositol 4,5-bisphosphate in the plasma membrane (step 4 ). Inositol trisphosphate, a water-soluble compound, diffuses from the plasma membrane to the endoplasmic reticulum, where it binds to specific IP3 receptors and causes Ca2_ channels within the ER to open. Sequestered Ca2_ is thus released into the cytosol (step 5), and the cytosolic [Ca2_] rises sharply to about 10_6 M. One effect of elevated [Ca2_] is the activation of protein kinase C (PKC). Diacylglycerol cooperates with Ca2_ in activating PKC, thus also acting as a second messenger (step 6 ). PKC phosphorylates Ser or Thr residues of specific target proteins, changing their catalytic activities (step 7). There are a number of isozymes of PKC, each with a characteristic tissue distribution, target protein specificity, and role. Gq proteins are class of G proteins which work to activate phospholipase C (PLC), participating in a variety of cellular signaling pathways, including taste, manic depression, tumor promotion, etc. The Gq protein works by activating PLC. PLC then cleaves a phospholipid. In the process, phosphatidylinositol 4,5-bisphosphate (PIP2) is cleaved into diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. IP3 then diffuses through the cytosol to bind to IP3 receptors, particular calcium channels in the endoplasmic reticulum (ER). These channels are specific to calcium and only allow the passage of calcium to move through. This causes the cytosolic concentration of calcium to increase,

causing a cascade of intracellular changes and activity. In addition, calcium and DAG together works to activate PKC, which goes on to phosphorylate other molecules, leading to altered cellular activity.

Catalytic:
The catalytic region or kinase core of the PKA allows for different functions to be processed; PKB (also known as Akt) and PKC kinases contains approximately 40% amino acid sequence similarity. This similarity increases to ~ 70% across PKCs and even higher when comparing within classes. For example, the two atypical PKC isoforms, and /, are 84% identical (Selbie et al., 1993). Of the over-30 protein kinase structures whose crystal structure has been revealed, all have the same basic organization. They are a bilobal structure with a sheet comprising the N-terminal lobe and a helix constituting the C-

terminal lobe. Both the ATP- and substrate-binding sites are located in the cleft formed by these two lobes. This is also where the pseudosubstrate domain of the regulatory region binds. Another feature of the PKC catalytic region that is essential to the viability of the kinase is its phosphorylation. The conventional and novel PKCs have three phosphorylation sites, termed: the activation loop, the turn motif, and the hydrophobic motif. The atypical PKCs are phosphorylated only on the activation loop and the turn motif. Phosphorylation of the hydrophobic motif is rendered unnecessary by the presence of a glutamic acid in place of a serine, which, as a negative charge, acts similar in manner to a phosphorylated residue. These phosphorylation events are essential for the activity of the enzyme, and 3phosphoinositide-dependent protein kinase-1 (PDK1) is the upstream kinase responsible for initiating the process by transphosphorylation of the activation loop.(Balendran et al., 2000) The consensus sequence of protein kinase C enzymes is similar to that of protein kinase A, since it contains basic amino acids close to the Ser/Thr to be phosphorylated. Their substrates are, e.g., MARCKS proteins, MAP kinase, transcription factor inhibitor IB, the vitamin D3 receptor VDR, Raf kinase, calpain, and the epidermal growth factor receptor.

Activation:
Upon activation, protein kinase C enzymes are translocated to the plasma membrane by RACK proteins (membrane-bound receptor for activated protein kinase C proteins). The protein kinase C enzymes are known for their long-term activation: They remain activated

after the original activation signal or the Ca2+-wave is gone. This is presumably achieved by the production of diacylglycerol from phosphatidylinositol by a phospholipase; fatty acids may also play a role in long-term activation.

Pathology:
Protein kinase C, activated by tumor promoter phorbol ester, may phosphorylate potent activators of transcription, and thereby lead to increased expression of oncogenes, promoting cancer progression, or interfere with other phenomena.

Conclusion:
Within the last decade the field of GPCR signaling has experienced an epiphany with the realization that GPCRs do more than subserve restricted functions in fully differentiated cells; they also play important roles in mediating diverse cell functions such as embryogenesis, tissue regeneration, and cell proliferation. Interestingly, this realization coincided with a similarly profound discovery in the field of asthma research that ASM not only contracts, but also performs numerous "synthetic" functions that modulate both airway structure and airway inflammation. Not surprisingly, ASM GPCRs are important regulators of many ASM synthetic functions.