Functional diversity of Ga subunits

The classification of G proteins is based on their amino acid identities and functional specialization of their Ga subunits (Simon et al. 1991). There are four major subfamilies of Ga subunits and their general properties are listed in Tables 4.1 and 4.4.

4.4.1 Gi subfamily

The largest subfamily is generally known as 'inhibitory' Ga subunits, after the identification of the negative regulator of AC, Ga;. All members except Gaz are subject to ADP-ribosylation mediated by the bacterial toxin from Bordetella pertussis (PTX), and such covalent modification prevents the G proteins from being activated by GPCRs. The three Gaj subtypes (Gaj1, Gaj2, and Gaj3) can all inhibit AC to a similar extent. Little is known about the functional differences between the three subtypes. A mutation at Arg179 constitutively activates Gaj2 and it is associated with adrenocarcinoma, ovarian cancer, and pituitary adenoma (Williamson etal. 1995). Mutation of Phe200 to Leu of Gaj2 leads to a pathological status known as idiopathic ventricular tachycardia (Lerman et al. 1998). Gaj2 is the most commonly used Gi subtype for studying Gi-linked receptors. Gaj2-deficient mice exhibit profound alterations of thymocyte maturation, elevated IgG levels in the large bowel, growth retardation and the development of lethal diffuse colitis with adenocarcinoma in the colon (Rudolph etal. 1995). Gaj3 is found in both plasma membrane and Golgi apparatus indicating that Gaj3 plays a role in membrane transport/remodelling processes. Gaj3-deficient cardiocytes derived from embryonic stem cells show inability to activate an inwardly rectifying K+ channel in response to muscarinic or adenosine receptor agonists (Sowell et al. 1997). The first regulator of G protein signalling identified in mammalian cells (GAIP) and nucleobindin (or Calnuc) show preferential interactions with Gaj3 (De Vries et al. 1995; Lin et al. 2000).

Gaz is often considered as the PTX-resistant alternative of Gaj, as it couples to most Gi-linked receptors and inhibit AC (Wong et al. 1992). Gaz is predominantly expressed in neuronal tissues (reviewed in Ho and Wong 2001). Specific molecular targets of Gaz include the Gaz-specific GAP, RGSZ1, G protein-regulated inducers of neurite outgrowth (GRIN1 and GRIN2) that are enriched in nerve growth cones, a GTPase-activating protein of monomeric G protein Rap1 (Rap1GAP), and the transcription regulator Eya2. Genetic deletion of Gaz impairs platelet aggregation by preventing the inhibition of cAMP formation by epinephrine (Yang et al. 2000). Gaz-deficient mice are more resistant to fatal thromboembolism, exhibit exaggerated response to cocaine, and have impaired responses to antidepressants that act as catecholamine reuptake inhibitors. The acute analgesic effect of morphine is also reduced, but another similar study indicates that deletion of Gaz facilitates the development of morphine tolerance (Hendry et al. 2000).

Two cDNA species of Gao have been isolated and they are splice variants of one single gene (Murtagh et al. 1994). A third form, GaoC, is derived from GaoA by deamidating the

Table 4.4 G protein-regulated effectors

Effector

Ga

GßY

Adenylyl cyclases

Type I

Gas f, Gai,o,z I,

Gß1 -3Y1 -2

(except Gß2Yi) 1

Type II

Gas f

Gß1 -3Y1 -2

(except Gß2Yi) f *

Type III

Gas,o|f f, Gai j

Type IV

Gas f

f *

Type V

Gas f, Gai z 1

Gßi Y2 f

Type VI

Gas f, Gai z ^

Gßi Y2 f

Type VII

Gas f

f *

Type VIII

Gas f, Gai ^

Type IX

Gas f, Gai 1

Arrestin

f

cGMP phosphodiesterases

Gat1,t2,gust f

No effect

Dynamin I

f

G protein receptor kinases

GRK1

No effect

GRK2

Gß1 —2 Y2

f

GRK3

Gß1 -3Y2

f

G protein-regulated inducers of neurite outgrowth GRIN1 Gao f

GRIN2 Gaio,z f

Ion channels

Ca2+, N-type

Gai1 'i2,z 1

Gßl,3 1

Ca2+, L-type

Gas f

Cl—, cardiac

Gas f

Cl—, epithelial

Gai,o f

Na+, cardiac

Gas f

Na+, epitheial

Gai3 1

GIRK1

Gai,z f

f

GIRK2

Gai,z f

f

GIRK4

Gai,z f

f

Monomeric G proteins

CDC25Mm

Gßi Y2, Gßi Y5, f

p140RasGRF

Gß1 Y2, Gß1 Y5, f

Raf-1

f

Shc

f

RaplGAP

Gai,o,z f

RaplGAPII

Gai,o,z f

p115RhoGEF

Ga13 f

PDZRhoGEF

Ga12,13 f

GAP1m

Gai2 f

Table 4.4 (continued)

Effector

Ga

GßY

Phospholipases

Type A2

t

Type C Pi

Gaq '11,14,16 t

Gß1Y1, Gß1Y2, Gß5Y2 t

Type C P2

Gaq-11,14,16 t

Gß1 Y1, Gß1 Y2' Gß5Y2' Gß3Y13

Type C P3

Gaq'11'14,16 t

Gß1Y1, Gß1Y2 t

Type C P4

Gaq'11,14,16 t

No effect

Type C e

Ga12 t

Phosphoinositide 3-kinases

Type P

t

Type y

Gao t

^Y^ Gß1-3Y2 t'

Plasma membrane Ca2+ pump

t

Transcription cofactor Eya2

Gai2,z I

Tyrosine kinases

Btk

Gaq,12 t

t

Tsk

t

Lck

Gas t

Lyn

Gaq t

Src

Gas, Gai2 t

t

Syk

Gaq 'Gai t, Gas |

* Effective only in the presence of active Gas or PKC-mediated phosphorylation.

Asn346 into Asp post-translationally (Exner etal. 1999). Gao is neural-specific, particularly concentrated in nerve growth cones, and constitutes about 1% of the total membrane proteins in neurons. The neuronal functions of Gao include inhibition of voltage-gated Ca2+ channels (Herlitze et al. 1996) and neuron-enriched type I AC (Taussig et al. 1994), as well as activation of mitogen-activated protein kinases (MAPKs; Moxham and Malbon, 1996). Gao is implicated in the development of nerve growth cones (Igarashi et al. 1993), vesicle trafficking (Gasman etal. 1997), and survival of primary accessory olfactory neuron (Tanaka etal. 1999). Like Gaz, Gao interacts with Rap1GAP (Jordon etal. 1999) and GRIN proteins (Chen et al. 1999). Other neuronal-specific molecules shown to associate with Gao include GAP-43 (Strittmatter etal. 1991), Alzheimer's disease gene presenilin-1 (Smine etal. 1998), Purkinje cell protein-2 (Luo and Denker 1999), amyloidogenic Ap-(1-40) and Ap-(25-35) (Rymer and Good 2001). Activated GaoA can induce transformation of NIH 3T3 cells via Src and STAT3, but not MAPK, thus providing a link between G protein and STAT-related signalling pathways (Ram etal. 2000).

Two transducin molecules (Gat1 and Gat2) and gustducin (Ggust) are the major regulators of cGMP phosphodiesterases. The three related Ga subunits have very restricted expression patterns. Gat1 are Gat2 are found only in rod and cone photoreceptor cells, whereas Ggust is found exclusively in the taste buds of all taste papillae, brush cells in the stomach and intestine, and the vomeronasal neuroepithelium. Subsequent detection of Gat1 in taste cells links its function to bitter taste perception (see Gilbertson et al. 2000). Enormous efforts have been directed toward Gat1 as a prototypical model for studying receptor-G protein-effector interactions, and detailed structures of rhodopsin (the primary receptor for Gat1),

Gt1 in various forms, and cGMP phosphodiesterase (partial structure) and other related molecules like RGS9 have been resolved (Hamm 1998; Palczewski et al. 2000; Slep et al. 2001). A mutation found in the first nucleotide binding region (Gly-38 to Asp) results in the 'Nougaret' form of congenital stationary night blindness (Dryja et al. 1996). Ggust is responsible for transducing both sweet and bitter tastes (Wong et al. 1996). Specific tastesensitive GPCRs have been cloned (Hoon et al. 1999). Co-localization of Ggust with Gß3 and Gy 13 suggests that they form a functional heterotrimer for regulating phosphodiesterase and PLCß2 in taste cells (Yan etal. 2001).

4.4.2 Gs subfamily

Gs subfamily members include two well-known ubiquitously expressed alternatively spliced variants of Gas, Gas(long) and Gas(short), the olfactory-specific Gaolf and an 'extra-large' Gas or XLGas which represents the third spliced variant of Gas. Amino acid sequence identities among the members of Gs subfamily are highest when compared with others subfamilies and they all stimulate ACs. Except Gaolf, all other Gas subtypes are actually splice variants of a single gene (GNAS).

A series of Gas mutations have been associated with various hormonal diseases and cancers (Weinstein and Yu 1999). Inactivating and activating mutations in the GNAS1 gene are associated with pseudohypoparathyroidism, type Ia and McCune-Albright syndrome, respectively. Regulation of the GNAS gene expression is of particular interest and it is actually an imprinted gene with the two alleles expressing different gene products. Pseudo-hypoparathyroidism type Ia is maternally inherited, which suggests that GNAS1 may be maternally imprinted. A differentially methylated locus containing an additional exon of GNAS1 has recently been identified. This exon is included within transcripts homologous to rat mRNAs encoding XLGas. In contrast to Gas-encoding transcripts, XLGas transcripts are derived from the paternal allele exclusively. The role of XLGas in the pathogenesis of pseudohypoparathyroidism requires further exploration.

In addition to adenylyl cyclase, Gas can positively regulate several types of ion channels (Table 4.4). Associations of Gas with different types of tyrosine kinases permit cross talks between GPCRs and growth factor receptors. Gas is activated by EGFR and Src kinase via tyrosine phosphorylation. Interestingly, Src kinase is also one of the downstream effectors of Gas (Ma etal. 2000). Gas represses adipogenesis in 3T3-L1 fibroblasts, and the process involves a direct interaction between Gas and a Src-like tyrosine kinase Syk, in which the phosphorylation of Syk is repressed (Wang and Malbon 1999). Induction of adipogenesis leads to a decline of Gas level and relieves Syk for phosphorylation. In contrast, Gas can stimulate another tyrosine kinase Lck directly in S49 lymphoma and subsequently leading to apoptosis (Gu et al. 2000). These observations suggest that Gas actively participates in the control of cell fate.

A family of putative odorant GPCRs exhibits restricted expressions in olfactory epithelium (Ronnett and Snyder 1992). Enhancement of both cAMP and phosphoinositide turnover has been demonstrated in response to odorants both in isolated olfactory cilia and in primary olfactory neuronal cultures. The Ga subunit involved in olfaction is Gaolf. Homozygous Gaolf -knockout mice show a striking reduction in the electrophysiologic response of primary olfactory sensory neurons to a wide variety of odors, but the topographic map of primary sensory projections to the olfactory bulb remains unaltered. The homozygotes are fertile, but exhibit hyperactive behaviours and inadequate maternal behaviours in mutant females.

Recently, dopamine Di and adenosine A2a receptors in the striatum are shown to be functionally associated with Gao]f (Corvol et al. 2001), suggesting that Gao]f is required for olfactory signal transduction and may function as a signalling molecule in the brain.

4.4.3 Gq subfamily

Gq subfamily members are regulators of phosphatidylinositol-specific PLCp isoforms and include Gaq, Ga11, Ga14, and Ga15/16. Gaq and Ga11 are found in most of the mammalian cells, while the other members show distinctive expression patterns. Gaq is the most extensively studied member and it activates PLCp in response to mitogenic signals such as bombesin and lysophosphatidate. Activation of Gq by type 1a metabotropic glutamate receptor induces phosphorylation of Gaq at Tyr-356, whereas Burton's tyrosine kinase (Btk) is one of the direct effectors of Gaq (Bence etal. 1997). Mice over-expressing wild-type and constitutively activated Gaq in the myocardium develop hypertrophy and heart failure associated with apoptosis. Expression of Gaq inhibitory peptides or RGS4 attenuates development of pressure overload hypertrophy. Gaq-deficient mice are viable but bear impaired cerebellar motor function and platelet aggregation. Ga11 is closely related to Gaq both structurally and functionally.

Ga14 is the least known member of the Gq subfamily in terms of its functional importance. Ga14 shows a distinctive receptor coupling profile, with marginal overlaps with Gi- and Gs-linked receptors (Ho et al. 2001). The receptor coupling promiscuity of Ga14 is significantly narrower than that of Ga15/16. Most of the Ga14-coupled receptors are expressed in various imflammatory cells where Ga14 is mainly expressed (Nakamura et al. 1991). However, the functions of these tissues remain intact in Ga14-knockout mice. An intriguing fact of Ga14 is that it may mediate an inhibitory effect on phosphoinositide metabolism in Xenopus oocytes ectopically expressing type 1 metabotropic glutamate receptor and Ga14 (Nakamura et al. 1994). The mechanism of this phenomenon is largely unknown.

Ga15 and Ga16 are the two orthologs cloned from mouse and human, respectively. They are promiscuous in terms of receptor coupling. Most GPCRs that normally interact with Gs or Gi can efficiently activate Ga15/16 and stimulate PLC^ (Offermanns and Simon 1995). In comparison with other Ga subunits, Ga15/16 have an extraordinarily long a4/^6 loop which is one of the major receptor-interacting regions of Ga. Changes in the lengths and identities of the C-terminal region (up to a4 helix) can alter the receptor coupling specificity ofGa16 (Mody etal. 2000). This region is also significantly different between Ga15 and Ga16, and it may contribute to the minor differences in their receptor coupling profiles. Ga15/16 are expressed mainly in hematopoietic cell lineages, and T cell receptor-CD3 complex can functionally activate both Gaq and Ga16 (Stanners et al. 1995; Zhou et al. 1998). Expression of activated mutant of Ga16 markedly suppresses the growth of small cell lung carcinoma through the activation of c-Jun N-terminal kinase (JNK) and Ca2+ responses (Heasley et al. 1996). Control of the stimulation of Ga16 in an optimal level appears to be essential for the induction of erythroid differentiation in MB-02 erythroleukemia cells (Ghose et al. 1999). Anaphylatoxin C5a receptor-mediated activation of PLC is reduced in macrophages of Ga15-knockout mice. However, there is no apparent hematopoietic defect in these knockout mice and they are viable and fertile.

Functional redundancy of Gq subfamily members have been implied in various trans-genic mice studies. Deletion of Gaq appears to produce the most severe physiological defects compared with other members. Significant overlaps of the defective consequences are observed in mice with both Gaq and one of the other members knocked out simultaneously.

Interestingly, other double-knockout mice with Can, Ga14, or Ga15/16 deleted do not have extensive defects (for transgenic mice studies, see Offermanns 2001).

4.4.4 G12 subfamily

The smallest subfamily of Ga subunits consists of only two members, Ga12 and Ga13. Their signalling characteristics are very diverse and distinct from the other subfamilies. Activated mutants of both Ga12 and Ga13 can trigger the activation of JNK in a Ras-dependent manner leading to apoptotic events (Prasad et al. 1995). The bridging molecule between Ga12 and Ras has been recently identified as GAP1m, a GTPase-activating protein of Ras, through the inspiration of the interaction between Ga12 and Btk (Jiang et al. 1998). Both GAP1m and Btk contain a novel sequence motif known as the Btk motif, which is an essential element for interacting with Ga12. Ga12- and Ga13-mediated activation of JNK appears to involve MEKK1, which phosphorylates and activates JNK, and apoptotic signal-regulating kinase 1 (Berestetskaya et al. 1998). Other studies also suggest that Ga12-regulated JNK activity is Rac-dependent (Collins et al. 1996). The Ras- and Rac-dependent pathways seem to work cooperatively in controlling cellular transformation (Tolkacheva et al. 1997). A novel form of PLC, PLCe, has been cloned recently and it acts as another links between Ga12 and Ras (Lopez etal. 2001). Ga12 directly activates PLCe and it serves as a nucleotide exchange factor of Ras to trigger Ras-dependent MAPK pathways.

Activation of Ga12 and Ga13 leads to extensive rearrangements of cytoskeleton with the induction of tyrosine phosphorylation of focal adhesion kinase, paxillin and p130 Crk-associated substrate in a Rho-dependent manner. Two nucleotide exchange factors of Rho, namely PDZRhoGEF and p115RhoGEF, have recently been shown to functionally associate with Ga12 and Ga13, respectively (Fukuhara etal. 1999; Hart etal. 1998). Although Ga12 can also bind p115RhoGEF, the complex is non-productive, indicating that the co-occurrence of specific types of G12 subfamily members and RhoGEF can determine the ultimate cell fate upon the actions of appropriate extracellular signals. Both RhoGEFs are more than bridging molecules because they also act as RGSs on their specific Ga partners (Kozasa et al. 1998). BothGa12 and Ga13 stimulate Na+/H+ exchange in PKC-dependent and independent manners, respectively. The Ga12-mediated pathway is also dependent on Ras and PLC. For Ga13, two streams of signals—one is Cdc42-MEKK1 related, another is Rho-dependent but MEKK1-independent—are responsible for the regulation of the activity of Na+/H+ exchanger NHE-1 (Hooley etal. 1996; Lin etal. 1996).

More recently, Ga12 and Ga13 have been linked to the regulation of cadherin/p-catenin signalling event. Both Ga subunits in their activated forms directly bind to the C-terminal cytoplasmic domains of N-cadherin, E-cadherin, and cadherin-14 (Meigs et al. 2001), and thus prevent the association of P-catenin to the same region. P-catenin dissociates and translocates into the nucleus to regulate transcription activity by interacting with adenomatous polyposis coli (APC) protein (Peifer and Polakis 2000).

An atypical Gah subunit was found to have the structure and functions resembling type II transglutaminase (Nakaoka et al. 1994). It has very low sequence identity with all other Ga subunits (<10% ) and a unique 16-amino acid guanine nucleotide-binding motif is exceptionally different from the five discrete nucleotide-binding pockets of the typical Ga subunits (Iismaa et al. 2000). Yet, it can directly stimulate phosphoinositide-dependent PLC81 upon the activation of a1-adrenoceptor subtypes (Baek et al. 2001).

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