Physiol. Rev. 85: 1159-1204, 2005;
doi:10.1152/physrev.00003.2005
0031-9333/05 $18.00
Mammalian G Proteins and Their Cell Type Specific Functions
Nina Wettschureck and
Stefan Offermanns
Institute of Pharmacology, University of Heidelberg, Heidelberg, Germany
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ABSTRACT
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Heterotrimeric G proteins are key players in transmembrane signaling by coupling a huge variety of receptors to channel proteins, enzymes, and other effector molecules. Multiple subforms of G proteins together with receptors, effectors, and various regulatory proteins represent the components of a highly versatile signal transduction system. G protein-mediated signaling is employed by virtually all cells in the mammalian organism and is centrally involved in diverse physiological functions such as perception of sensory information, modulation of synaptic transmission, hormone release and actions, regulation of cell contraction and migration, or cell growth and differentiation. In this review, some of the functions of heterotrimeric G proteins in defined cells and tissues are described.
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I. INTRODUCTION
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All cells possess transmembrane signaling systems that allow them to receive information from extracellular stimuli like hormones, neurotransmitters, or sensory stimuli. This fundamental process allows cells to communicate with each other. All transmembrane signaling systems share two basic elements, a receptor which is able to recognize an extracellular stimulus as well as an effector which is controlled by the receptor and which can generate an intracellular signal. Many transmembrane signaling systems like receptor tyrosine kinases incorporate these two elements in one molecule. In contrast, the G protein-mediated signaling system is relatively complex consisting of a receptor, a heterotrimeric G protein, and an effector. This modular design of the G protein-mediated signaling system allows convergence and divergence at the interfaces of receptor and G protein as well as of G protein and effector. In addition, each component, the receptor, the G protein as well as the effector can be regulated independently by additional proteins, soluble mediators, or on the transcriptional level. The relatively complex organization of the G protein-mediated transmembrane signaling system provides the basis for a huge variety of transmembrane signaling pathways that are tailored to serve particular functions in distinct cell types. It is probably this versatility of the G protein-mediated signaling system that has made it by far the most often employed transmembrane signaling mechanism. In this review we summarize some of the biological roles of G protein-mediated signaling processes in the mammalian organism which are based on their cell type-specific function. Although we have tried to cover a wide variety of cellular systems and functions, the plethora of available data forced us to restrict this review. Particular emphasis is placed on cellular G protein functions that have been studied in primary cells or in the context of the whole organism using genetic approaches.
A. Basic Principles of G Protein-Mediated Signaling
More than 1,000 G protein-coupled receptors (GPCRs) are encoded in mammalian genomes. While most of them code for sensory receptors like taste or olfactory receptors, 
400–500 of them recognize nonsensory ligands like hormones, neurotransmitters, or paracrine factors (53, 185, 519, 534, 649). For more than 200 GPCRs, the physiological ligands are known (Table 1). GPCRs for which no endogenous ligand has been found are "orphan" GPCRs (376, 389, 688).
Upon activation of a receptor by, e.g., its endogenous ligand, coupling of the activated receptor to the heterotrimeric G protein is facilitated. Multiple site-directed mutagenesis experiments have been performed on G protein-coupled receptors, and they have revealed various cytoplasmic domains of the receptors that are involved in the specific interaction between the receptors and the G protein. However, despite the determination of the structure of rhodopsin at atomic resolution (504), it is still not clear how specificity of the receptor-G protein interaction is achieved and how a ligand-induced conformational change in the receptor molecule results in G protein activation (177, 212, 213, 565, 674).
The heterotrimeric G protein consists of an 
-subunit that binds and hydrolyzes GTP as well as of a 
- and a 
-subunit that form an undissociable complex (233, 255, 475). Several subtypes of -, -, and -subunits have been described (Table 2). To dynamically couple activated receptors to effectors, the heterotrimeric G protein undergoes an activation-inactivation cycle (Fig. 1). In the basal state, the -complex and the GDP-bound -subunit are associated, and the heterotrimer can be recognized by an appropriate activated receptor. Coupling of the activated receptor to the heterotrimer promotes the exchange of GDP for GTP on the G protein -subunit. The GTP-bound -subunit dissociates from the activated receptor as well as from the -complex, and both the -subunit and the -complex are now free to modulate the activity of a variety of effectors like ion channels or enzymes. Signaling is terminated by the hydrolysis of GTP by the GTPase activity, which is inherent to the G protein -subunit. The resulting GDP-bound -subunit reassociates with the -complex to enter a new cycle if activated receptors are present. For recent excellent reviews on basic structural and functional aspects of G proteins, see References 49, 83, 361, and 526.
While the kinetics of G protein activation through GPCRs has been well described for quite a while, only recently has the regulation of the deactivation process been understood in more detail. Based on the observation that the GTPase activity of isolated G proteins is much lower than that observed under physiological conditions, the existence of mechanisms that accelerate the GTPase activity had been postulated. Various effectors have indeed been found to enhance GTPase activity of the G protein -subunit, thereby contributing to the deactivation and allowing for rapid modulation of G protein-mediated signaling (23, 45, 348, 571). More recently, a family of proteins called "regulators of G protein signaling" (RGS proteins) has been identified, which is also able to increase the GTPase activity of G protein -subunits (272, 481, 550). There are 30 RGS proteins currently known, which have selectivities for G protein -subfamilies. The physiological role of RGS proteins is currently under investigation. Besides their role in the modulation of G protein-mediated signaling kinetics, they also influence the specificity of the signaling process and in some cases may have effector functions.
The interaction of G proteins with the inner side of the plasma membrane is facilitated by lipid modifications of both the -subunit as well as of the -subunit of the -complex (97, 448, 589, 728). Recent data provide evidence that heterotrimeric G proteins of the Gi family are also involved in receptor-independent processes (52, 413), which appear to be critically involved in the positioning of the mitotic spindle and the attachment of microtubules to the cell cortex (234). These processes also involve a group of proteins that carry a so-called GoLoco motif which functions as a guanine nucleotide dissociation inhibitor (684).
B. G Protein -Subunits and -Complexes
The functional versatility of the G protein-mediated signaling system is based on its modular architecture and on the fact that there are numerous subtypes of G proteins. The -subunits that define the basic properties of a heterotrimeric G protein can be divided into four families, Gs, Gi/Go, Gq/G11, and G12/G13 (Table 2). Each family consists of various members that often show very specific expression patterns. Members of one family are structurally similar and often share some of their functional properties. The -complex of mammalian G proteins is assembled from a repertoire of 5 G protein -subunits and 12 -subunits (Table 2). While 1- to 4-subunits form a tight complex with -subunits which can only be separated under denaturing conditions, the 5-subunit interaction with -subunits is comparably weak (347, 543). The 5-subunit is an exception in that it can also be found in a complex with a subgroup of RGS proteins (689). The -complex was initially regarded as a more passive partner of the G protein -subunit. However, it has become clear that -complexes freed from the G protein -subunit can regulate various effectors (112). These -mediated signaling events include the regulation of ion channels (488), of particular isoforms of adenylyl cyclase and phospholipase C (169, 615), as well as of phosphoinositide-3-kinase isoforms (641). With a few exceptions, the ability of different -combinations to regulate effector functions does not dramatically differ (112).
Most receptors are able to activate more than one G protein subtype. The activation of a G protein-coupled receptor therefore usually results in the activation of several signal transduction cascades via G protein -subunits as well as through the freed -complex. The pattern of G proteins activated by a given receptor determines the cellular and biological response, and activated receptors that lead to functionally similar or identical cellular effects usually activate the same G protein subtypes. The G protein receptor interaction in general does not occur in an absolutely specific or in a completely promiscuous manner. Some receptors appear to interact only with certain G protein subforms, and in some cellular systems, the compositions of defined G protein-mediated signaling pathways can be very specific. However, there are some characteristic patterns of receptor-G protein coupling that have been described for the majority of receptors (Fig. 2).
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FIG. 2. Typical patterns of receptor/G protein coupling. Although there are many exceptions, three basic patterns of receptor-G protein coupling have been found which critically define the cellular response after ligand-dependent receptor activation. 2, 2-adrenergic receptor; D1–5, dopamine receptor subtypes 1 to 5; GIRK, G protein-regulated inward rectifier potassium channel; 5-HT1,2, serotonin receptor subtypes 1 and 2; M1–5, muscarinic acetylcholine receptor subtypes 1 to 5; mGluR1–7, metabotropic glutamate receptor subtypes 1 to 7; PLC-, phospholipase C-; PI-3-K, phosphoinositide-3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; Rho-GEF, Rho-guanine nucleotide exchange factor; TP, thromboxane A2 receptor; IP, prostacyclin receptor.
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The G proteins of the Gi/Go family are widely expressed and especially the -subunits of Gi1, Gi2 and Gi3 have been shown to mediate receptor-dependent inhibition of various types of adenylyl cyclases (615). Because the expression levels of Gi and Go are relatively high, their receptor-dependent activation results in the release of relatively high amounts of -complexes. Activation of Gi/Go is therefore believed to be the major coupling mechanism that results in the activation of -mediated signaling processes (112, 543). The function of members of the Gi/Go family has often been studied using a toxin from Clostridium botulinum (pertussis toxin; PTX) which is able to ADP-ribosylate most of the members of the Gi/Go family close to their COOH termini. COOH-terminally ADP-ribosylated Gi/Go is unable to interact with the receptor. Thus PTX treatment results in the uncoupling of the receptor and Gi/Go. The structural similarity between the 3 Gi subforms suggests that they may have partially overlapping functions. In contrast to other G proteins, the effects of Go, which is particularly abundant in the nervous system, appears to be primarily mediated by its -complex. Whether Go can regulate effectors directly is currently not clear. A less widely expressed member of the Gi/Go family is Gz (438), which in contrast to Gi and Go is not a substrate for PTX. Gz is expressed in various tissues including the nervous system and platelets. It shares some functional similarities with Gi-type G proteins but has recently been shown to interact specifically with various other proteins including Rap1GAP and certain RGS proteins (438). Several -subunits like gustducin and transducins belong to the Gi/Go family and are involved in specific sensory functions (24, 126).
The Gq/G11 family of G proteins couples receptors to -isoforms of phospholipase C (169, 538). The -subunits of Gq and G11 are almost ubiquitously expressed while the other members of this family like G14 and G15/16 (G15 being the murine, G16 the human ortholog) show a rather restricted expression pattern. Receptors that are able to couple to the Gq/G11 family do not appear to discriminate between Gq and G11 (490, 660, 696, 705). Similarly, there is obviously no difference between the abilities of both G protein -subunits to regulate phospholipase C -isoforms. While Gq and G11 both are good activators of 1-, 3-, and 4-isoforms of phospholipase C (PLC), the PLC 2-isoform is a poor effector for both (538). The biological significance of the diversity among the Gq gene family is currently not clear. While the importance of Gq and G11 in various biological processes has been well established, the roles of G14 and G15/16, which show very specific expression patterns, are not clear. Mice carrying inactivating mutations of the G14 and G15 genes have no or very minor phenotypical changes (132; H. Jiang and M. I. Simon, personal communication). In contrast, mice lacking Gq or both Gq and G11 have multiple defects (489, 494, 495) (see below).
The G proteins G12 and G13, which are often activated by receptors coupling to Gq/G11, constitute the G12/G13 family and are expressed ubiquitously (139, 607). The analysis of cellular signaling processes regulated through G12 and G13 has been difficult since specific inhibitors of these G proteins are not available. In addition, G12/G13-coupled receptors usually also activate other G proteins. Most information on the cellular functions regulated by G12/G13 therefore came from indirect experiments employing constitutively active mutants of G12/G13. These studies showed that G12/G13 can induce a variety of signaling pathways leading to the activation of various downstream effectors including phospholipase A2, Na+/H+ exchanger, or c-jun NH2-terminal kinase (139, 193, 276, 523, 607). Another important cellular function of G12/G13 is their ability to regulate the formation of actomyosin-based structures and to modulate their contractility by increasing the activity of the small GTPase RhoA (79). Activation of RhoA by G12 and G13 is mediated by a subgroup of guanine nucleotide exchange factors (GEFs) for Rho which include p115-RhoGEF, PDZ-RhoGEF, and LARG (194, 236, 618). While the RhoGEF activity of PDZ-RhoGEF and LARG appears to be activated by both G12 and G13, p115-RhoGEF activity is stimulated only by G13. Recently, an interesting link between G12/G13 and cadherin-mediated signaling was described, both G12 and G13 interact with the cytoplasmic domain of some type I and type II class cadherins, causing the release of -catenin from cadherins (434, 435). Various other proteins including Bruton's tyrosine kinase, the Ras GTPase-activating protein Gap1m, radixin, heat shock protein 90, AKAP110, protein phosphatase type 5, or Hax-1 have also been shown to interact with G12 and/or G13 (309, 359, 485, 532, 638, 709).
The ubiquitously expressed G protein Gs couples many receptors to adenylyl cyclase and mediates receptor-dependent adenylyl cyclase activation resulting in increases in the intracellular cAMP concentration. The -subunit of Gs, Gs, is encoded by GNAS, a complex imprinted gene that gives rise to several gene products due to the presence of various promoters and splice variants (Fig. 3). In addition to Gs, two transcripts encoding XLs and Nesp55 are generated by promoters upstream of the Gs promoter. While the chromogranin-like protein Nesp55 is structurally and functionally not related to Gs, XLs is structurally identical to Gs but has an extra long NH2-terminal extension that is encoded by a specific first exon (329). In contrast to Gs, XLs has a limited expression pattern being mainly expressed in the adrenal gland, heart, pancreatic islets, brain, and the pars intermedia of the pituitary (509). However, XLs shares with Gs the ability to bind to -subunits and to mediate receptor-dependent stimulation of cAMP production (33, 339). Interestingly, the first exon of the Gnasxl gene encodes another protein termed ALEX (338), which is able to interact with the XL domain of XLs and to inhibit its activity (188, 338). Interestingly, Nesp55 and XLs are differentially imprinted. While the promoter of Nesp55 is DNA-methylated on the paternally inherited allele resulting in the expression only from the maternally inherited allele, the promoter driving XLs expression is methylated on the maternal allele, and XLs is only expressed from the paternal allele (244, 245, 515). Several other transcripts like Nespas of which some are believed to be untranslated show ubiquitous expression and are derived from the paternal allele due to differentially methylated promoter regions (243, 294, 396, 619). In contrast, the promoter driving the expression of Gs has been shown to be biallelically active and to lack differential methylation (85, 244, 245, 733). However, in a few tissues such as the renal proximal tubules, the thyroid, pituitary, and ovaries, the paternal Gs expression is silenced by an as yet undefined mechanism (209, 242, 394, 414, 723).
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II. CARDIOVASCULAR SYSTEM
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A. Autonomic Control of Heart Function
Cardiac regulation by the sympathetic system is mediated by -adrenergic receptors that are coupled primarily to Gs (Fig. 4). cAMP produced in response to Gs activation directly modulates the gating of hyperpolarization-activated, cyclic nucleotide-gated channels and activates protein kinase A (PKA) which in turn phosphorylates several proteins involved in excitation-contraction coupling including L-type Ca2+ channels, phospholamban, or troponin I (44). These cellular changes are believed to underlie the well-known effects of sympathetic cardiac activation including positive chronotropic, dromotropic, lusitropic, and inotropic effects (545). Transgenic overexpression of the short form of Gs (Gs-S) in the murine heart had no effect on the basal cardiac function but resulted in an enhanced efficacy of -adrenoceptor Gs signaling, and chronotropic and inotropic responses to catecholamines were increased (299). Once Gs-overexpressing mice become older, they develop clinical and pathological signs of cardiomyopathy (300). These pathological processes are accompanied by a lack of normal heart rate variability as well as of protective desensitization mechanisms (635, 650). The development of cardiomyopathy after prolonged overexpression of Gs is in line with the current concept of the pathophysiological mechanisms underlying the development of chronic heart failure. The insufficient cardiac output characteristic for heart failure typically goes along with an increased sympathetic tone resulting in chronic catecholamine stimulation of cardiomyocytes, which is believed to be deleterious (71). Although the 1-adrenoceptor is the predominant subtype expressed in cardiomyocytes, also 2-adrenoceptors are expressed in the heart (545). Interestingly, there is increasing evidence that 1- and 2-adrenoceptors play different roles in catecholamine-induced cardiomyopathy. Mice overexpressing human 2-adrenoceptors have only slightly altered cardiac function and appear to have normal life expectancy (259, 260, 443, 544) while mice overexpressing 1-adrenoceptors develop severe hypertrophy and die of heart failure (162). The 1- and 2-adrenoceptors also differ with regard to their signal transduction. While 1-adrenergic receptors are Gs coupled, 2-adrenoceptors are also able to couple to Gi-type G proteins (700, 701) (Fig. 4). The additional activation of Gi via 2-adrenergic receptors may explain the observed differences in signaling induced via 1- and 2-adrenergic receptors (116, 125, 232, 405, 725, 736). This led to the hypothesis that 2-adrenoceptor stimulation exerts some sort of protection against cardiac hypertrophy and failure, especially under conditions of chronic activation of the -adrenergic system and that this is due to signaling via Gi. The well-documented upregulation of Gi in human heart failure (174, 482) may be a mechanism to counteract deleterious Gs-mediated signaling. The potential cardioprotective role of Gi is also supported by studies in mice. While the overexpression of 2-adrenergic receptors in normal cardiomyocytes is well tolerated, mice which lack in addition the major Gi -subunit, Gi2, die within a few days after birth (180). Mice that overexpress 2-adrenoceptors in cardiomyocytes and which carry only one intact Gi2 gene allele develop more pronounced cardiac hypertrophy and earlier heart failure compared with 2-adrenoceptor transgenic animals with normal Gi2 levels.
The muscarinic acetylcholine (M2) receptor that is coupled to Gi/Go G proteins mediates the parasympathetic regulation of the heart (Fig. 4). The negative chronotropic and dromotropic effects of the parasympathetic system are believed to result from the Gi-mediated inhibition of adenylyl cyclase, resulting in an inhibition of the cAMP production as well as by the activation of G protein-regulated inward rectifier potassium channels (GIRK) by -subunits released from activated Gi/Go (601). The atrial GIRK consists of Kir3.1 and Kir3.4 subunits. Mice lacking either of the two channel subunits have normal basal heart rates but show reduced vagal and adenosine-mediated slowing of heart rate and markedly reduced heart rate variability, which is thought to be determined by the vagal tone (47, 680). The involvement of G complexes in regulation of GIRK channels has been well established using electrophysiological and biochemical approaches (349, 398, 679). Mice in which the amount of functional G protein was reduced by more than 50% in cardiomyocytes also show an impaired parasympathetic heart rate control (207). The central role of Gi in inhibitory regulation of heart rate and atrioventricular conductance has led to attempts to treat cardiac arrhythmias by atrioventricular nodal gene transfer of Gi2 in a model of persistent atrial fibrillation in swine (146). While wild-type Gi2 did not change basal heart rate, a constitutively active mutant of Gi2 resulted in a significant decrease in heart rate. When tested for their effects in a model for tachycardia-induced cardiomyopathy, the condition was significantly improved by wild-type Gi2 and even more by constitutively active Gi2 (37). In addition to the stimulatory regulation of potassium channels, muscarinic regulation of heart function also involves inhibition of voltage-dependent L-type Ca2+ channels via an unknown mechanism. In mice lacking the -subunit of Go, inhibitory muscarinic regulation of cardiac L-type Ca2+ channels was abrogated, although Go represents only a minor fraction of all G proteins in the heart (639). Interestingly, mice which lack the -subunit of Gi2 (Gi2) also show a severely affected inhibitory regulation of L-type Ca2+ channels via muscarinic M2 receptors (101, 468). This suggests that both G proteins, Go and Gi2, are involved in the regulation of cardiac L-type Ca2+ channels.
B. Myocardial Hypertrophy
Myocardial hypertrophy is the chronic adaptive response of the heart to injury or increased hemodynamic load. It is characterized by increased cardiomyocyte size and protein content, as well as altered gene expression, recapitulating an embryonic phenotype (109, 301). Such pathological myocardial hypertrophy was shown to be associated with increased cardiac mortality (191, 285, 535), raising the question whether prevention of pathological hypertrophy is beneficial or not (191). Several mechanosensitive mechanisms involving stretch-activated ion channels, integrins or Z-disc proteins were suggested to mediate myocardial hypertrophy in response to pressure overload (191, 285, 535). In addition, GPCR agonists like norepinephrine/phenylephrine, angiotensin II, or endothelin-1 were shown to induce a hypertrophic phenotype in cultured rat embryonic cardiomyocytes (4, 341, 560, 581). These ligands are known to activate Gq/G11-coupled receptors, such as the 1-adrenergic receptor, the angiotensin AT1 receptor, or the endothelin ETA receptor (362, 561, 592). Activation of Gq by Pasteurella multocida toxin (559) or expression of wild-type Gq (5, 362) induces the hypertrophic phenotype in cultured cardiomyocytes, while inhibition of Gq/G11 by the RGS domain of GRK2 inhibited agonist-induced hypertrophy (423). In vivo, cardiac-restricted expression of wild-type (128) or constitutively active Gq (437) results in cardiac hypertrophy. In addition, in vivo overexpression of typically Gq/G11-coupled receptors (444, 474) or their downstream effectors (65, 454, 656) induces hypertrophy. Conversely, in vivo inhibition of Gq/G11 by overexpression of RGS4, a GTPase-activating G protein for Gq/G11 and Gi/Go (547), or by overexpression of the COOH terminus of Gq (10) results in a reduced hypertrophic response, and cardiomyocyte-specific inactivation of the genes encoding Gq/G11 completely abrogates the hypertrophic response elicited by pressure overload (677). Interestingly, an impaired hypertrophic response due to inhibition of Gq/G11-mediated signaling does not negatively influence long-term cardiac function (166), suggesting that hypertrophy in response to pressure overload is not necessarily required to maintain cardiac function. In addition to pressure overload-induced myocardial hypertrophy, the Gq/G11-mediated signaling pathway was also implicated in the pathogenesis of diabetic cardiomyopathy. Gq levels and PKC activity were shown to be enhanced in the streptozotocin-induced diabetic rat heart (714), and heart specific overexpression of RGS4 protected mice against different models of diabetic cardiomyopathy. In contrast, heart-specific expression of a RGS-resistant Gq caused sensitization towards diabetic cardiomyopathy (235). The downstream signaling processes in Gq/G11-mediated hypertrophy are complex and not fully understood (Fig. 5). Intracellular Ca2+ mobilization in response to activation of Gq/G11-coupled receptors promotes Ca2+/calmodulin (CaM)-dependent activation of calcineurin, which in turn mediates dephosphorylation and nuclear translocation of transcription factors of the NFAT (nuclear factor of activated T cells) family. Although activation of the calcineurin/NFAT signaling pathway is clearly sufficient to induce myocardial hypertrophy, it is not completely clear whether inhibition of this signaling pathway prevents hypertrophy (for review, see Refs. 190, 191). In addition, a variety of other effectors have been implicated in myocardial hypertrophy, such as protein kinase C (PKC) isoforms, mitogen-activated protein (MAP) kinases, the phosphatidylinositol (PI) 3-kinase/Akt/GSK-3 pathway or small GTPases (for review, see Refs. 148, 191, 285, 535).
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FIG. 5. Gq/G11 family G proteins are centrally involved in myocardial hypertrophy. G protein-coupled receptors like the 1-adrenergic receptor (1), the angiotensin AT1 receptor, or the endothelin ETA receptor act through Gq/G11 to induce hypertrophy via activation of downstream effectors including the calcineurin/NFAT pathway, PKC isoforms, MAP kinases, the PI-3-kinase/Akt/GSK-3 pathway or small GTPases. CaM, calmodulin; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinases; NFAT, nuclear factor of activated T cells; PI-3-K, phosphoinositide-3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC-, phospholipase C-.
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GPCRs known to mediate myocardial hypertrophy can also activate G12/G13 family G proteins, resulting in the activation of RhoA (215, 257). RhoA was suggested to be involved in the hypertrophic responses to phenylephrine, endothelin, chronic hypertension, or overexpression of Gq (128, 264, 278, 360, 562, 567). Expression of inhibitory COOH-terminal peptides of G12 and G13, as well as expression of the G12/G13 specific RGS domain of p115RhoGEF, inhibited phenylephrine-mediated JNK activation in neonatal cardiomyocytes (423). In addition, overexpression of a constitutively active mutant of G13 induced a hypertrophic response in neonatal cardiomyocytes, with increased expression of the hypertrophy-associated embryonic gene program (179). However, no in vivo data on the role of G12/G13 in myocardial hypertrophy are available.
C. Smooth Muscle Tone
Smooth muscle tone is controlled by the phosphorylation state of the regulatory light chain (MLC20) of myosin II (for review, see Refs. 269, 593, 594). MLC20 is phosphorylated by the Ca2+/CaM-dependent myosin light chain kinase (MLCK), leading to enhanced velocity and force of actomyosin cross-bridging. Dephosphorylation of MLC20 is mediated by myosin phosphatase, an enzyme that is negatively regulated by the Rho/Rho-kinase pathway. Thus increased contractility can be achieved through Ca2+-mediated MLCK activation and through Rho-dependent inhibition of MLC20 dephosphorylation. A variety of transmitters and hormones regulate smooth muscle tone through GPCRs (Fig. 6). Typical vasoconstrictor receptors, such as the angiotensin AT1 receptor, the endothelin ETA receptor, or the 1-adrenergic receptor, act on Gq/G11-coupled receptors (159, 215, 724) to enhance intracellular Ca2+ concentration, leading to MLCK activation. Increased intracellular Ca2+ levels are not only due to IP3-mediated Ca2+ release from the sarcoplasmic reticulum, but also to Ca2+ influx through cation channels or voltage-gated Ca2+ channels (for review, see Refs. 269, 593). In addition, many Gq/G11-coupled receptors have been shown to activate RhoA, thereby contributing to Ca2+-independent smooth muscle contraction (593, 594). Smooth muscle specific overexpression of a COOH-terminal Gq peptide, which is believed to inhibit the receptor/G protein interaction, ameliorates hypertension induced by long-term treatment with phenylephrine, serotonin, or angiotensin II (331). Mice lacking RGS2, a GTPase activating G protein which accelerates the inactivation of Gq/G11, suffer from hypertension (261). Interestingly, it was recently shown that the nitric oxide/cGMP cascade, which constitutes the main relaxant pathway in smooth muscle cells, negatively regulates Gq/G11 signaling by cGMP kinase-mediated phosphorylation and activation of RGS2 (625). However, in addition to this peripheral vascular mechanism, an increased sympathetic tone might contribute to elevated arterial blood pressure in RGS2-deficient mice (227).
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FIG. 6. Heterotrimeric G proteins involved in the regulation of smooth muscle tone. Gq/G11-coupled receptors increase the intracellular Ca2+ concentration, leading to Ca2+/calmodulin (CaM)-dependent MLCK activation and MLC20 phosphorylation. Especially G12/G13-coupled receptors mediate RhoA activation, thereby contributing to Ca2+-independent smooth muscle contraction. Relaxation is induced by activation of Gs-coupled receptors, but the mechanisms underlying cAMP-mediated relaxation are not clear. Gi-mediated signaling might contribute to contraction by inhibiting Gs-mediated relaxation. cGKI, cGMP-dependent protein kinase I; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; MLC20, regulatory chain of myosin II; MLCK, myosin light-chain kinase; MPP, myosin phosphatase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, cAMP-dependent kinase; PLC-, phospholipase C-; RhoGEF, Rho specific guanine nucleotide exchange factor; ROCK, Rho kinase; TRP, transient receptor potential channel.
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In vitro, most Gq/G11-coupled vasoconstrictor receptors also activate G12/G13 family G proteins, like the receptors for endothelin-1, vasopressin, angiotensin II (215, 257), thrombin (411), or thromboxane A2 (491, 492). Constitutively active forms of G12 and G13 induced a pronounced, RhoA-dependent contraction in cultured vascular smooth muscle cells, and receptor-mediated contractions were strongly inhibited by dominant negative forms of G12 and G13 (215). These data suggest that also the G12/G13-mediated signaling pathway is involved in the regulation of smooth muscle tone, most likely by modulating the activity of myosin phosphatase via Rho/Rho-kinase. In accordance with this, inhibition of Rho-kinase was shown to normalize blood pressure in humans and experimental animals (426, 636). The relative contribution of Gq/11/Ca2+-mediated and G12/13/Rho/Rho-kinase-mediated signaling to regulation of vascular smooth muscle tone is not clear. However, data obtained in visceral smooth muscle suggested that Gq/G11 conveys a fast, transient response, while G12/G13 mediates a sustained, tonic contraction (257).
Vascular smooth muscle relaxation is mediated by a variety of mechanisms, one of them being the activation of Gs-coupled receptors like the adenosine A2 receptors, 2-adrenergic receptors, or prostaglandin receptor subtypes IP, DP, and EP2. How the subsequent increase in cAMP levels reduces smooth muscle tone is not understood. In vitro data suggest that the relaxant effect is partially due to a PKA-mediated MLCK phosphorylation, which decreases the enzyme's affinity for the Ca2+/CaM complex, but the physiological relevance of this signaling pathway is unclear. Possible other substrates for PKA are heat shock protein 20, RhoA, or myosin phosphatase (for review, see Ref. 269). cAMP has also been suggested to cross-activate cGMP kinase I in vascular or airway smooth muscle (32, 390), but this hypothesis has been questioned by the finding that vessels from cGKI-deficient mice relax normally in response to cAMP (518). In addition, cAMP-independent mechanisms of Gs-mediated relaxation involving large-conductance, Ca2+-activated K+ (MaxiK, BK) channels have been proposed (624).
The role of Gi-mediated signaling in vascular smooth muscle tone seems to differ between different vessel types. An inhibitory effect of PTX on norepinephrine-induced contractility was reported in rat tail artery (516, 600) but was absent in aorta (517). High blood pressure in spontaneously hypertensive rats is preceded by increased expression of Gi proteins (18, 19), and PTX treatment delayed the onset of hypertension (387), suggesting that decreased cAMP levels play a role in the pathogenesis of this model of hypertension. Enhanced signaling via a PTX-sensitive G protein was reported in immortalized B lymphoblasts from patients with essential hypertension (520), and this was attributed to a C825T polymorphism in the gene coding for G3, a constituent of the Gi heterotrimer (586). The C825T polymorphism was suggested to be associated with an increased risk of hypertension, obesity, and arteriosclerosis in some (for review, see Ref. 585) but not in all studies (283, 616, 617). However, the significance of genetic association studies in general remains controversial (20, 199, 291).
Very similar to vascular smooth muscle, also airway smooth muscle tone is mainly regulated by Gq/G11 family G proteins, which mediate bronchoconstriction, and Gs family G proteins, which mediate bronchorelaxation. Acetylcholine released from postganglionic parasympathetic nerves controls resting tone mainly via the Gq/G11-coupled M3 receptor subtype (90), but also other Gq/G11-coupled receptors are expressed in airway smooth muscles, like the H1 histamine receptor (133, 222), the leukotriene CysLT1 receptor (314), the B2 bradykinin receptor (421, 630), the ETB endothelin receptor (216, 241, 441), and others. Airway hyperreactivity in the A/J mouse strain was suggested to be due to enhanced agonist affinity and increased G protein coupling efficiency of the M3 muscarinic receptor (205), and Gq protein was shown to be upregulated in antigen-induced airway hyperresponsive rats (106). Mice lacking the -subunit of Gq showed impaired metacholine-induced airway responses and lacked the typical increase in metacholine sensitivity after allergen sensitization and reexposition (55). Not much is known about the role of G12 and G13 in airway smooth muscle tone regulation. The fact that repetitive antigen challenge significantly increases the expression of these proteins in airway smooth muscle suggests a role in allergic asthma (105, 108), but direct evidence for an involvement of G12/G13 is still lacking. Gs-coupled receptors play an important role in the relaxation of contracted airway smooth muscle, most prominently the 2-adrenergic receptor, but also the prostaglandin E2 receptor EP2 (512) or the prostacyclin IP receptor (41) (for review, see Ref. 628). The Gi family of G proteins contributes to the regulation of airway smooth muscle contractility mainly by inhibiting the relaxant effects of Gs. The inhibitory effect of PTX on acetylcholine-induced bronchoconstriction is negligible in normal rats, but significant in rats suffering from antigen-induced airway hyperresponsiveness (107). In these mice, Gi3 protein is upregulated in bronchial smooth muscle cells, suggesting that the relative contribution of Gi-mediated constriction is increased in antigen-challenged airway smooth muscle (107).
D. Platelet Activation
Platelets are small cell fragments that circulate in the blood and adhere at places of vascular injury to the vessel wall where they become activated resulting in the formation of a platelet plug that is responsible for primary hemostasis. Platelets can also become activated under pathological conditions, e.g., on ruptured atherosclerotic plaques leading to arterial thrombosis. Platelet adhesion and activation is initiated by their interaction with adhesive macromolecules like collagen and von Willebrand factor (vWF) at the subendothelial surface (303, 554). While collagen is able to induce firm adhesion of platelets to the subendothelium (666), the recruitment of additional platelets to the growing platelet plaque requires the local accumulation of diffusible mediators that are produced or released once platelet adhesion has been initiated, and some level of activation through platelet adhesion receptors has occurred (3). These mediators include ADP/ATP and thromboxane A2 (TxA2), which are secreted or released from activated platelets as well as thrombin, which is produced on the surface of activated platelets. These platelet stimuli have in common their action through G protein-coupled receptors. While ADP induces the activation of Gq and Gi via P2Y1 and P2Y12 receptors (197, 354), the activated TxA2 receptor (TP) couples to Gq and G12/G13 (337, 492) (Fig. 7). G protein-coupled protease-activated receptors (PARs) that are activated by thrombin are functionally coupled to Gq, G12/G13, and in some cases to Gi (121). In response to these secondary mediators of platelet activation, platelets immediately undergo a shape change reaction during which they become spherical and extrude pseudopodia-like structures. In addition, the glycoprotein IIb/IIIa (integrin IIb3) undergoes a conformational change resulting in binding of fibrinogen/vWF and subsequent platelet aggregation. Finally, the formation and release of TxA2, thrombin, and ADP is further stimulated. Thus secondary mediators increase through G protein-coupled receptors their own formation resulting in an amplification of their effects, and eventually all G protein-mediated signaling pathways induced via these receptors become activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. Progress has recently been made using genetic mouse models in understanding the role of individual G protein-mediated signaling pathways during platelet activation.
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FIG. 7. Role of heterotrimeric G proteins in mediating platelet activation by soluble mediators like ADP, thromboxane A2 (TxA2), thrombin, and epinephrine. Major roles are played by the G proteins Gq, G13, and Gi which couple receptors to the indicated effector molecules. The subsequent signaling processes eventually lead to platelet responses like shape change, degranulation, and aggregation (for details, see text). TP, TxA2 receptor; PAR, protease-activated receptor; P2Y1/P2Y12, purinergic receptors; 2A, 2A-adrenergic receptor; RhoGEF, Rho guanine nucleotide exchange factor; PLC-2/3, phospholipase C-2/3; PI-3-K, phosphoinositide-3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; PIP3, phosphatidylinositol 3,4,5-trisphosphate.
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The requirement of Gq-mediated signaling for agonist-induced platelet activation has been demonstrated by the phenotype of Gq-deficient platelets, which fail to aggregate and to secrete in response to thrombin, ADP, and TxA2 due to a lack of agonist-induced phospholipase C activation. This dramatic phenotype found in Gq-deficient platelets is due to the fact that platelets lack G11 (313), which is in most other cells coexpressed with Gq and can compensate Gq deficiency. Mice lacking Gq have increased bleeding times and are protected against collagen/epinephrine-induced thromboembolism (494). Although Gq-mediated signaling appears to be absolutely required for platelet activation, there is clear evidence that also Gi type G proteins need to be activated to induce full activation of integrin IIb3. In mice lacking the -subunit of Gi2, the response of ADP that acts through the Gi-coupled P2Y12 receptor is reduced (304). However, also the effects of mediators like thrombin and TxA2, which primarily signal through Gq and G12/G13 were found to be inhibited in platelets lacking Gi2 (304, 712). This supports the view that platelet activation by thrombin and thromboxane A2 requires in part the action of secondary mediators like ADP, which are released after activation of Gq-mediated signaling pathways through TxA2 and thrombin receptors. An important role of the Gi-mediated signaling pathway in platelet activation is also suggested by studies in platelets lacking the Gq-coupled P2Y1 receptor or after pharmacological blockade of P2Y1 (170, 251, 310, 379, 568). These platelets do not aggregate in response to low and intermediate concentrations of ADP unless Gq-mediated signaling is induced via activation of another receptor. Similarly, platelets lacking P2Y12 or in which P2Y12 was pharmacologically blocked did not aggregate in response to ADP unless the Gi-mediated pathway was activated via a different receptor (181, 568). Thus there is clear evidence that Gq and Gi synergize to induce platelet activation. It is currently not clear how Gi contributes to integrin IIb3 activation in platelets, but a decrease in cAMP levels is unlikely to be involved (129, 529, 569, 712). Another member of the Gi family of heterotrimeric G proteins, Gz, has been implicated in platelet activation induced by epinephrine acting on 2-adrenergic receptors. In contrast to ADP, TxA2, and thrombin, epinephrine is alone not able to fully activate mouse platelets. However, it is able to potentiate the effect of other platelet stimuli. In Gz-deficient platelets, the inhibitory effect of epinephrine on adenylyl cyclase and epinephrine-potentiating effects were strongly impaired while the effects of other platelet activators appear to be unaffected (713).
Despite the central role of Gq in platelet activation, it was recently demonstrated that induction of Gi- and G12/G13-mediated signaling pathways is sufficient to induce integrin IIb3 activation (149, 483). Interestingly, in G13-deficient platelets, but not in G12-deficient platelets, the potency of various stimuli including TxA2, thrombin, and collagen to induce platelet shape change and aggregation is markedly reduced (455). These defects are accompanied by a defect in the activation of RhoA and a delayed phosphorylation of the myosin light chain as well as by an inability to form stable platelet thrombi under high sheer stress conditions (455). In addition, mice carrying platelets that lack G13 have an increased bleeding time and are protected against the formation of arterial thrombi induced in a carotid artery thrombosis model (455). These data indicate that in addition to Gq and Gi also G13 is crucially involved in the signaling processes mediating platelet activation via G protein-coupled receptors both in hemostasis and thrombosis. These findings also indicated that G13-mediated signaling is not only involved in the response of platelets to relatively low stimulus concentrations that induce platelet shape change but is also required for normal responsiveness of platelets at higher stimulus concentrations. A reduced potency of platelet activators in the absence of G13-mediated signaling becomes in particular limiting under high flow conditions that lead to a rapid clearance of soluble stimuli from the site of platelet activation and formation of mediators. In addition, the defective activation of RhoA-mediated signaling in the absence of G13 appears to contribute to the observed defect in the stabilization of platelet aggregates under high sheer stress ex vivo as well as in vivo. In fact, RhoA-mediated signaling has been suggested to be required for platelet aggregation under high sheer conditions as well as for the irreversible aggregation of platelets in suspension (450, 570).
These studies have clearly shown that three G proteins are major mediators of platelet activation via G protein-coupled receptors: Gq, Gi2, and G13. However, even in the absence of either Gq, Gi2, or G13 some platelet activation can still be induced, while in the absence of both Gq and G13, platelets are unresponsive to thrombin, TxA2, or ADP. This indicates that the activation of Gi-mediated signaling alone is not sufficient to induce any platelet activation (456). The optimal activation of platelets under physiological and pathological conditions obviously requires the parallel signaling through several heterotrimeric G proteins.
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III. ENDOCRINE SYSTEM AND METABOLISM
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The endocrine system consists of a variety of glands and other structures that produce, store, and secrete hormones directly into the systemic circulation, thereby controlling electrolyte and water homeostasis, metabolism, growth, reproduction, etc. GPCRs contribute to endocrine functions in a twofold way: 1) by mediating hormonal end organ effects and 2) by controlling hormone secretion itself. Hormone secretion, as well as secretion from neuronal or exocrine cells, typically involves elevation of cytosolic Ca2+ and/or cAMP (for review, see Refs. 160, 738). In most secretory cells, Ca2+ influx through voltage-operated Ca2+ channels is the dominant mode of regulation, like in adrenal chromaffin cells (160), while in other cells, such as anterior pituitary gonadotropes, Ca2+ mobilization from internal stores is the critical step (633). In yet another endocrine cell, such as lactotroph cells, both increased intracellular Ca2+ levels and cAMP production contribute to secretion (130, 186, 620). Accordingly, with rare exceptions, activation of Gs and/or Gq/G11 family G proteins enhances secretion regardless of the endocrine cell type involved.
A. Hypothalamo-Pituitary System
Hormone release from the anterior pituitary is tightly controlled by hypothalamic releasing hormones and release inhibiting factors, all of which act through GPCRs. The receptors for corticotropin-releasing hormone (263) and growth hormone-releasing hormone (GHRH) (588) primarily act through Gs, while receptors for gonadotropin-releasing hormone (445), thyrotropin-releasing hormone (211, 720), and the many prolactin-releasing factors (186) mainly act through Gq/G11 family G proteins, and only partly through Gs. In addition to their secretagogue effects, hypothalamic releasing hormones regulate hormone synthesis and cell proliferation (81, 430, 531, 583, 587, 647). Anterior pituitary secretion and proliferation is not only stimulated by the classical hypothalamic releasing hormones, but also by a variety of other factors, such as the gastrointestinal peptide hormone ghrelin, which enhances growth hormone (GH) secretion via the predominantly Gq/G11-coupled growth hormone secretagogue receptor GHS-R (343, 588), or members of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily, which exert secretagogue effects on a variety of pituitary cell types via their Gs-coupled receptors (579). The in vivo relevance of Gs family G proteins in anterior pituitary function was studied in mice and in patients with inactivating or activating Gs mutants. Somatotroph-specific overexpression of cholera toxin, which irreversibly activates Gs by ADP ribosylation, caused somatotroph hyperplasia, increased GH levels and gigantism in mice (82). In humans, activating mutations of GNAS can be found in 40% of GH producing pituitary tumors (363, 406), as well as in 10% of nonfunctioning pituitary adenomas (406, 632, 685). These activating mutations of GNAS encode substitutions of either Arg-201 or Gln-227, two residues that are critical for the GTPase reaction (187, 223, 363, 406). In GH-secreting tumors, the mutation is almost always in the maternal allele, presumably because Gs is mainly expressed from the maternal allele ("paternally imprinted") in pituitary cells (242). Activating GNAS mutations were also, though rarely, found in corticotroph (541, 686), but not in thyrotroph tumors (147, 406). Such activating somatic GNAS mutations are not necessarily restricted to the pituitary, but are often part of the McCune-Albright syndrome, which is defined by the trias fibrous dysplasia of bone, café-au-lait skin pigmentation, and endocrine hyperfunctions of variable degree (for review, see Refs. 599, 669). Endocrine hyperfunction is due to constitutive activation of Gs signaling in other endocrine glands, leading to adrenal hyperplasia with Cushing syndrome (60, 182), precocious puberty (138, 574), or hyperthyroidism (see sect. IIIC). In melanocytes, increased Gs activity mimics the activity of melanocyte stimulating hormone, leading to typical café-au-lait hyperpigmentation (334).
Heterozygous inactivating GNAS mutations result in Albright hereditary osteodystrophy (AHO), a congenital disorder characterized by obesity, short stature, brachydactyly, subcutaneous ossifications, and neurobehavioral deficits of variable severity (for review, see Refs. 13, 366, 599, 669). In addition to these defects, patients with maternally inherited mutations show multihormone resistance (termed pseudohypoparathyroidism type Ia, PHP1a) in tissues with a paternally imprinted GNAS allele, such as proximal tubules of the kidney, thyroid, or ovaries (209, 242, 414, 723). In these tissues, the effects of Gs-coupled hormone receptors, like those for parathyroid hormone, thyroid stimulating hormones, or the gonadotropins, are impaired. Clinically, this results in variable degrees of hypocalcemia and hyperphosphatemia, hypothyroidism (see also sect. IIIC), and delayed or incomplete sexual development and reproductive dysfunction in women (13, 366, 599, 669). These abnormalities of the reproductive system are easily explained by malfunction of receptors for follicle-stimulating hormone and luteinizing hormone. In addition, at least in mice, the Gs-coupled orphan receptor GPR3 is crucially involved in the maintenance of meiotic arrest in oocytes (432, 433).
The phenotype of humans heterozygous for an inactivating GNAS mutation is partly reproduced in mice carrying a targeted disruption of Gnas exon 2. In these animals, PTH resistance was only found if the mutation was maternally inherited, and only these animals showed reduced Gs expression in the renal cortex (723). In humans, renal PTH resistance without Albright hereditary osteodystrophy (PHPIb) can also be due to other GNAS mutations, such as a mutant which results in a biallelic paternal imprinting phenotype (395), or a mutant unable to interact with the PTH receptor (697). Yet another GNAS mutation causes impaired signaling via the PTH and TSH receptors, but enhanced signaling via the likewise Gs-coupled receptor for luteinizing hormone, leading to enhanced testosterone production. This paradoxical combination of gain and loss of function is explained by the fact that the underlying GNAS mutation results in a constitutively active form of Gs which, however, is temperature sensitive. The mutant is stable only at the relatively lo
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