G protein-coupled receptor
G-protein coupled receptors (GPCRs) are integral plasma membrane proteins that transduce signals from extracellular 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 extracellular signal is typically amplified to produce robust, varied, and cell-specific responses.
GPCRs are quite distinct from growth-factor receptors (e.g. insulin, NGF, BDNF, ...), which signal through tyrosine kinases, do not use heterotrimeric G proteins, and typically regulate gene expression. However, GPCRs and growth-factor receptors do share several common final pathways of signaling.
The GPCR gene family is one of the largest gene families with more than 800 homologous genes in man (Foord et al., 2005). In the central nervous system, the GPCRs include receptors for many neurotransmitters and hormones and for transduction of several sensory inputs, as well as receptors for chemokines  and wnt signaling. Especially for discussion of neurotransmitter receptors, neurobiologists like to call the GPCRs "metabotropic receptors" (implying coupling to metabolism) to distinguish them from the ligand-gated ion channels of fast chemical synapses, which they call "ionotropic." Common GPCR ligands in the nervous system are:
- Monoamines: adrenaline, noradrenaline, serotonin, dopamine, histamine
- Other small neurotransmitters: Acetylcholine (mACh), gamma aminobutyric acid (GABAB), glutamate (metabotropic, mGluR), ATP (P2Y), adenosine, cannabinoids. (Where the neurotransmitter has multiple classes of receptors, the receptor that is a GPCR is named in parentheses.)
- Many peptide neurotransmitters and hormones: opioids, somatostatin, NPY, oxytocin, vasopressin, neurotensins, VIP, galanin, kinins, releasing hormones, and more.
- Sensory modalities: light (rhodopsin), odorants, some tastetants including sweet, bitter, and umami
For most of these 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 and noradrenaline. Three of them couple to the G-protein Gq , often making calcium signals (α1 adrenergic receptors), three of them couple to Gi, often inhibiting adenylyl cyclase, activating GIRK channels, or inhibiting Ca channels (α2 adrenergic receptors), and three of them couple to Gs, often stimulating adenylyl cyclase (β 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.
On the basis of sequence and structural similarities, GPCRs can be divided into clans A, B, C, etc. or classes 1, 2, 3, etc.(Foord et al., 2005). The classes important for the nervous system and their ligands include:
- Class 1 or A (the very large rhodopsin family): all the ligands listed above except the few itemized below.
- Class 2 or B (the secretin family): secretin, VIP, PACAP, GHRH
- Class 3 or C (the glutamate family): mGluR, GABAB, CaSR (calcium-sensing), a few possible taste receptors
- Class F (the frizzled family): frizzled (the wnt receptor)
Signaling from GPCRs
GPCRs couple to GTP-binding heterotrimeric G proteins  that consist of Gα, Gβ, and Gγ subunits ( Figure 1). At rest these three subunits are assembled into a complex, Gαβγ. Since Gβ and Gγ are inseparable once coassembled, it is customary to talk about a resting GαGβγ complex. Typically, both Gα and Gβγ are held at the inner leaflet of the plasma membrane by hydrophobic lipid modifications. Unlike the schematic drawing in Figure 1, the heterotrimeric G protein is physically larger than the GPCR (typical molecular weights: Gαβγ ~90K, GPCRs, ~50K). The heterotrimeric G proteins should be distinguished from the small, low molecular weight, monomeric G proteins of the Ras, Rab, Rho, etc. families with which the Gα subunit does share some homology. The monomeric G proteins have the GTP-binding and GTPase properties of Gα subunit of heterotrimeric G proteins but they do not couple to GPCRs or Gβγ, and they signal to different downstream effectors.
Gα subunits are flexible signaling proteins. In the inactive resting complex, the Gα 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 Gα 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 Gα-GTP-Gβγ complex is unstable so that the active Gα-GTP and Gβγ separate from one another and from the receptor as well, but they usually remain attached to the plasma membrane by their lipid anchors. Thus there are now three active products that couple to downstream effectors: the activated receptor, Gα-GTP and the Gβγ dimer. 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 Gβγ dimer may leave the membrane and go into the cytoplasm or to other membranes. 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 Gα-GTP and Gβγ separately.
Downstream coupling of Gα-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 α subunit they contain, and there are nearly 20 genes encoding Gα subunits. However, for understanding much of their signaling it suffices to consider five broad Gα families and five signaling pathways that they most typically activate:
- Gαs , Gαolf : activates plasma membrane adenylyl cyclases, increasing cellular cyclic AMP (cAMP), which e.g. stimulates phosphorylation of target proteins by cAMP-dependent protein kinase. Gαs and its downstream signaling can be covalently activated by cholera toxin.
- Gαi , Gαo : inhibit most adenylyl cyclases, decreasing cellular cAMP. Gαi and Gαo can be covalently inactivated and their signaling turned off by Pertussis toxin. Gαo is said to constitute 1% of brain proteins. Being so abundant, the PTX-sensitive G proteins are the principal source of active Gβγ subunits.
- Gαq , Gα11 : activate phospholipase Cβ (PLCβ), which cleaves certain phosphoinositide lipids (PIP2) of the plasma membrane, and generates several second messengers e.g. IP3 that releases Ca2+ from intracellular stores and diacylglycerol that activates phosphorylation by protein kinase C.
- Gα12 , Gα13 : enhance Rho kinase and change expression of some genes and the phosphorylation of myosin
- Gαtransducin, Gαgustducin: activate cyclic GMP (cGMP) phosphodiesterase (transducin) that cleaves and depletes cytoplasmic cGMP (retina only) or cAMP phosphodiesterase (gustducin) that cleaves and depletes cAMP (some 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 Gβγ
The Gβγ subunits also are potent signals. They bind to several effectors. They activate G-protein coupled inwardly rectifying K+ (GIRK) channels. They inhibit opening of voltage-gated 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 Gβγ dimers act directly on at least two more downstream effectors: they stimulate PLC β and phosphoinositide 3-kinase γ (PI3Kγ).
Although there are numerous Gβ and Gγ genes, to a first approximation, the Gβγ populations associated with all Gα subunits are similar. By mass action, the strength of Gβγ signaling to effectors is probably greatest when launched by the most abundant Gα subunits (e.g. Go in neurons) or when derived from types of Gα subunits that dissociate most readily from their Gβγ partners.
Termination of GPCR signaling
Termination of signaling requires turning off activated receptors, turning off activated G-proteins, 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 over-stimulation. In one canonical shutdown pathway (Gainetdinov et al., 2004), 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 [[arrestins] ] at the plasma membrane. The arrestin-receptor complex may be unable to couple to downstream G proteins, but it may mediate other signaling 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 Gα-GTP is terminated by GTP hydrolysis, a reaction catalyzed by the Gα subunit itself that yields the inactive form, Gα-GDP. Thus activated G-proteins have an intrinsic self-timer function that terminates their activity. Gα-GDP in turn is a scavenger that binds any free Gβγ dimers, re-forming the inactive heterotrimeric G protein GαβγGDP. 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. For example, PLC is a powerful GAP for Gq-GTP (Ross, 2011). 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 (Hollinger and Hepler, 2002).
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 (non-classical signaling, Sun et al., 2007). 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 Gα 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.
- 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 gonadotropes.
- 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.
- 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.
- Finally significant convergence can be desirable. It allows diverse cells and organs to call for a common 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-or-fight, by glucagon from the pancreas to aid in energy distribution, or by histamine from mast cells to accompany allergic reactions.
Thus, although it is common to call nerve-released agonists neurotransmitters independent of 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: 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) in a paracrine, hormone-like manner rather than providing specific information to one neuron.
- GPCR actions take 100 ms to minutes. Fast chemical synapses signal in a fraction of a millisecond.
- 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.
- The GPCR coupled monoamines and peptides have longer extracellular lifetimes and thus cannot be targeted for point-to-point wiring to a single postsynaptic cell in a circuit. They work on larger groups of cells.
StructureAll GPCRs have a membrane topology with seven α-helical transmembrane segments, N-terminus outside and C-terminus inside ( Figure 1). X-ray crystal structures, available so far for several dozen GPCRs, show that the transmembrane segments are broken helices that cross the membrane at various angles ( Figure 2). With this many structures, common structural features can be deduced (Venkatakrishnan et al., 2013).
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 N-terminus. Biochemical experiments and crystal structures (Rassmussen et al., 2011) 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 ( Figure 3). 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 (Angers et al., 2002; Pin et al., 2006). 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 (Pin et al., 2006).
A time line
- 1878-1905 Langley postulates receptor concept for action of muscarine
- 1900-1950 Pharmacology of agonists and antagonists distinguishes many receptor subtypes phenomenologically
- 1950-1965 Concept of the first second messenger (cAMP) formulated by Sutherland as an intermediate in adrenaline and glucagon action (Nobel Prize)
- 1970-1980 Rodbell and Gilman recognize that a GTP binding protein conveys the receptor signal to adenylyl cyclase (Nobel Prize)
- 1986-1990 Initial cloning reveals that many pharmacological receptors are heptahelical like rhodopsin, and the common features of coupling to G-proteins inspire the concept of GPCRs as a receptor class with a uniform paradigm of signaling. Rhodopsin is considered the prototype member of this class.
- 2000-2013 Crystal structures of >20 GPCRs are published (Nobel Prize, Kobilka and Lefkowitz)
Angers S, Salahpour A, Bouvier M. (2002) Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annu Rev Pharmacol Toxicol. 42:409-35.
Foord SM, Bonner TI, Neubig RR, Rosser EM, Pin JP, Davenport AP, Spedding M, Harmar AJ. 2005. International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol Rev. 57:279-88.
Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, Caron MG. (2004) Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev Neurosci. 27:107-44.
Hollinger S, Hepler JR. (2002) Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev. 54:527-59.
Pin JP, Neubig R, Bouvier M, Devi L, Filizola M, Javitch JA, Lohse MJ, Milligan G, Palczewski K, Parmentier M, Spedding M. (2007) International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers. Pharmacol Rev. 59:5-13.
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Ross EM. (2011) Gαq and phospholipase C-β: turn on, turn off, and do it fast. Sci Signal. 4(159):pe5. doi: 10.1126/scisignal.2001798.
Sun Y, McGarrigle D, Huang XY. (2007) When a G protein-coupled receptor does not couple to a G protein. Mol Biosyst. 3:849-54.
Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM. (2013) Molecular signatures of G-protein-coupled receptors. Nature. 494(7436):185-94. doi: 10.1038/nature11896.
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