Direct Downstream Effectors Of The G Protein Pathway

5.1. Adenylyl Cyclases

Adenylyl cyclase (AC), a membrane protein, is important in promoting the conversion of adenosine triphosphate (ATP) to cAMP. Nine membrane-bound isoforms of AC and two spliced variants of AC8 have been cloned and characterized in mammals, and all are expressed in the brain (76). cAMP is an important second messenger in the cell. It stimulates the activation of PKA by binding to the regulatory subunit of PKA and removing the inhibition by that subunit.

PKA can then phosphorylate several substrates important in glucose metabolism and other vital physiological processes. cAMP can also interact with many other proteins to promote protein-protein interactions that are independent of PKA signaling. For example, cAMP can directly bind a RasGEF called Epac and stimulate its GEF activity toward the small G protein Rapl (77,78).

Several G proteins modulate AC activity directly. Gas, Gaj, and GPy can interact physically with various ACs to affect their activity. In particular, Gas can activate all nine isoforms of ACs to various degrees, whereas the Gaj family can inhibit some ACs, especially AC5 through AC6 (25,76).

Fig. 4. The Ga12/13 signaling pathways. This schematic diagram demonstrates how the Ga12/13 pathway regulates multiple cellular pathways to affect physiological processes such as transcription, proliferation, and cell movement. LPA, lysophosphatidic acid ; NHE, sodium-hydrogen exchanger; GEF, guanine exchange factor; Ras, a small GTPase; Rho, a small GTPase; PLD, phospholipase D.

Fig. 4. The Ga12/13 signaling pathways. This schematic diagram demonstrates how the Ga12/13 pathway regulates multiple cellular pathways to affect physiological processes such as transcription, proliferation, and cell movement. LPA, lysophosphatidic acid ; NHE, sodium-hydrogen exchanger; GEF, guanine exchange factor; Ras, a small GTPase; Rho, a small GTPase; PLD, phospholipase D.

The py-subunit has also been shown to be able to activate or inhibit some AC isoforms, depending on the py combination or on the presence of activated Gas. For example, Gp1y2 can inhibit AC1 and AC3, but some py complexes can directly activate AC2, AC4, and AC7 in the presence of activated Gas (12,76,79).

AC has also been shown to be important in numerous processes, such as long-term potentiation (LTP) and long-term memory, cell differentiation, development, and drug dependence (76,79). In some of these processes, the AC isoform involved is either activated or inhibited by calcium. For example, AC1 activity is stimulated by Ca2+/camodulin, and this AC isoform is important in LTP and long-term memory (76). In relation to GPCRs and Gas activation, AC in which Gas is coupled to P1-ARs functions in increasing cardiac rate and force of contraction; Gas coupled to P2-ARs functions in smooth muscle relaxation; Gas that is coupled to P3-ARs functions in lipolysis of white adipose tissue (80).

5.2. Phospholipase C

Various G proteins can activate phospholipases. Phospholipases such as phospholipase C (PLC) can cleave the polar head group of inositol phospholipids. All members of the Gaq family can activate PLC-P 1 to P4 isoforms; Ga12 can activate PLC-e; GPy can activate all four PLC-P isoforms as well as PLA2 (12). In particular, PLC-P can hydrolyze the phosphorylated lipid PIP2 to generate two intracellular products: inositol 1,4,5-trisphosphate (a universal calcium-mobilizing second messenger) and DAG (an activator of PKC). These products are important in raising intracellular Ca2+ levels in the cells and in activating Ca2+-sensitive proteins such as calmodulin, which regulates other proteins within the cell.

PLC-P is composed of the N-terminal pleckstrin homology (PH) domain, EF-hand domain, catalytic X and Y domains, and regulatory C2 domain

(81). The PH domains of PLC-P2 and PLC-P3 bind the heterotrimeric G protein subunit GPy. The C2 domains of PLC-P 1 and PLC-P2 bind the GTP-bound Gaq. Comparison of the ligand-binding affinities of different PLC-P isozymes shows that each isozyme is regulated differently by both subunits of Gaq. PLC-P2 and PLC-P3 are more sensitive to the Py-subunit than PLC-P1 and PLC-P4, whereas the affinities of Gaq for PLC-P 1 and PLC-P3 are higher than that for PLC-P2 (81).

Another phospholipase family member that can be modulated by G proteins is PLC-e. PLC-e can function as a phospholipase as well as a RasGEF

(82). It can interact with both large and small G proteins. To do so, PLC-e contains conserved catalytic and regulatory domains common to other eukaryotic PLCs, but it also contains two Ras-associating domains and a RasGEF motif. This isoform can hydrolyze PIP2, and this activity is selectively stimulated by a constitutively active form of Ga12 as well as various GPy-dimers (82,83). Additionally, PLC-e's lipase activity can be inhibited by pertussis toxin, suggesting that Ga/Gao signaling may be involved (84). Furthermore, PLC-e can promote formation of Ras-GTP through its RasGEF domain. However, it has been suggested that PLC-e is a Ras effector because the Ras-associating domain of PLC-e can bind to the H-Ras in a GTP-dependent manner that correlates with stimulation of PLC-e's lipase activity of PLC-e (85,86).

5.3. Ion Channels

Several ion channels are directly affected by G protein activation. GIRK, voltage-gated Ca2+ channels (VDCCs), cardiac and epithelial chloride (Cl) channels, and cardiac and epithelial sodium channels (Na+) are affected by G proteins. Such interactions have been deduced on the basis of several cri teria. The channel activity must be conditionally and reversibly modified by the activation of a relevant GPCR. The use of a nonhydrolyzable GTP analog should allow for channel modulation, even in the absence of receptor stimulation. The addition of a purified and active G protein subunit should be sufficient to trigger changes in channel activity. Finally, physical association between the G protein and the channel subunits must be demonstrated within the confines of an intact cell.

The most well-characterized channel that is directly affected by G protein activation is the GIRK, or Kir3, family. Direct activation of GIRKs by G proteins is involved in the rapid inhibition of membrane excitability resulting in the slowing of heart rate by the vagus nerve or the autoinhibitory release of dopamine by midbrain neurons. Therefore, these GIRKs couple GPCR signaling to membrane excitability. There are four isoforms of Kir3s that are affected by G proteins: Kir3.1 (GIRK1), Kir3.2 (GIRK2), Kir3.3 (GIRK3), and Kir3.4 (GIRK4). They are all expressed in the brain. GIRK4 is also expressed in the heart. The Kir3s were the first effectors demonstrated to be directly activated by the Gpy-subunit (75). The current view is that the interaction of the Gpy-subunits with the GIRK channel involves multiple binding domains that synergistically control channel gating. Several studies have been conducted using fusion proteins, mutagenesis, and peptides to identify the sites that are important for GIRK activity (87). Gpy directly binds to both the carboxy- and amino- cytoplasmic segments of the channel protein. It is also believed that Gpy binding stabilizes channel-PIP2 interactions that open the channel gate (88).

VDCCs are another type of ion channels that are directly affected by G protein signaling. These channels, located near vesicle docking sites, are important because they are responsible for the influx of Ca2+ into the presynaptic neuron, which allows for the release of neurotransmitters from the synaptic nerve terminals. Calcium ions act in concert with distinct components of the presynaptic machinery to facilitate fusion of synaptic vesicles within the plasma membrane. Modulating the entry of Ca2+ into the nerve terminal thus represents a major means by which neurotransmitter secretion can be controlled. There are four major families of VDCCs: N-type, L-type, P/Q-type, and T-type. The N-type Ca2+ (Ca2.2v) and L-type Ca2+ (Cav3.1) channels are regulated by G proteins. Gai1/i2/z and Gpy have been shown to inhibit the N-type Ca2+ channels, whereas Gas and Gpy can stimulate the L-type Ca2+ channel (89-93). Of these, the Gpy-complex has been shown to directly interact with and modulate N-type Ca2+ channel activity (89). In fact, the Gpy-complex can bind several contact sites on the a1p-subunit of the N-type Ca2+ channel. The interaction between Gpy- and a1p-subunits has been mapped to the N-terminal, C-terminal, and I-II loop of the a1p-subunit of the N-type Ca2+ channel (89).

5.5. Regulators of Small GTPases

Several small G protein regulators are direct effectors of G proteins. Ras-GRF (also known as CDC25Mm), Raf-1, and Shc are believed to be modulated by the GPy-complex, whereas Gapm and RhoGEFs are affected by the Ga-subunit (68,94-100). Of these, Raf-1 is believed to directly interact with the GPy-complex, whereas RapGAP and RhoGEFs are known to directly interact with the Ga-subunit.

The serine/threonine (Ser/Thr) protein kinase Raf-1, directly downstream of Ras, has been reported to interact with the GP2-subunit in vitro and in vivo (96). In competition assays, only P-AR kinase can inhibit GP2 binding to Raf-1 but can not inhibit Ras or 14-3-3. The significance of this interaction has not been determined, although overexpression of GP1y2 in HEK293 cells can stimulate the phosphorylation of mitogen-activated protein kinase (MAPK) and enhance the MAP/ERK kinase (MEK) kinase activity of c-Raf (101). These studies were performed in cell lines in which these proteins were overexpressed; therefore, the relevance of this interaction and the ki-nase assays need to be further investigated.

RasGRF has GEF activity toward the small G proteins Ras and Rac (95,102,103). RasGRF can be activated via two different pathways. The GPy-complex has been implicated in one of these pathways. Carbachol-treated fibroblasts transfected with the muscarinic receptor type 1 (Gaq-coupled) or type 2 (Garcoupled) increased the phosphorylation state of RasGRF as well as its GEF activity toward Ras. This increase can also be observed in COS-1 cells transfected with Gp1y2 complex, suggesting that it is the GPy-complex that is significant in activating RasGRF. Additionally, carbachol treatment of neonatal rat brain explants increased RasGRF's GEF activity and phosphorylation state (94). Furthermore, RasGRF immunopre-cipitated from HEK293 cells overexpressing GPy showed enhanced GEF activity toward the Rac1 protein (95).

The Ga12 family can also modulate the activity of the small G proteins Rho and Ras via their direct interaction with RhoGEF and RasGAP (99,100). Ga13 can physically interact with three RhoGEFs (p115PhoGEF, PDZORhoGEF, and LARG) and stimulate their guanine exchange activity (99,104,105). In contrast, Ga12 has been shown to physically interact with Gap1m, a RasGAP, via the PH-BM domain of RasGAP in vitro and in vivo. This interaction can stimulate the GAP activity of Gap1mtoward Ras (100).

Both Gai and Gao can modulate the small G protein Rap via binding to RapGAP directly (33,106). In particular, Ga; binds the Rap1GAPII isoform directly in its N-terminus. The activated form of Gai binds RaplGAPII more efficiently than wild-type Gai, and stimulation of Gi-coupled receptors recruits RapIGAPII from the cytosol to the membrane, which then leads to a decrease in the levels of RapGTP. Consequently, the decrease in Rapl leads to the activation of the MAPK, because there is less of Rap1 to inhibit Ras activity (106). In contrast, wild-type Gao can bind RaplGAPII directly and inhibit RapIGapII from acting on Rapl (33). Consequently, the increased levels of Rapl can stimulate MAPK1/2 activity. The mechanism by which this occurs can be attributed to the promotion of the ubiquination and protease degradation of RaplGAP upon binding to Gao (107,108).

Other small G proteins affected by G protein signaling include Rac and Cdc42. The activity of Rac can be modulated by Gaq signaling because Gaq-deficient platelets cannot activate Rac upon stimulation of thromboxane A2 receptor using the agonist U46619 (109). Constitutively active Gaq can stimulate Cdc42 activity toward insulin signaling to GLUT4 translocation in adipoctyes (110).

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