Interactions between G proteins and GPCRs

G proteins bind to their corresponding GPCRs at multiple sites on the cytoplasmic regions of the receptor. Several regions of Ga contribute toward association with GPCRs, which have been extensively reviewed (Gudermann etal. 1997; Bourne 1997; Hamm 1998). One of the major domains for receptor recognition is the C-terminus of Ga, where PTX-mediated ADP-ribosylation of Gi subfamily members occurs and the receptor-G protein interaction is then disrupted. Replacement of the last five amino acids of Gaq with those of Gaj2, Gao, Gaz, or Gas broadens the profile of receptor coupling (Conklin et al. 1993,1996). A similar strategy applied to studying the extraordinary receptor coupling property of promiscuous Ga16 indicates that the C-terminus of Ga contributes to the determination of receptor coupling specificity (Mody et al. 2000). A peptide derived from the last 11 residues of Gat1 impeded rhodopsin-mediated activation of Gat1 (Hamm et al. 1988). Using a photo-activatable crosslinking reagent, the third intracellular loop of rhodopsin is crosslinked with the residues 342-345 of the extreme C-terminus of Gat1 (Cai et al. 2001), which clearly demonstrated the direct contact between the C-terminal tail of Ga and the cognate receptor.

GaoA and GaoB have identical C-terminal tails but they couple to different receptors to mediate the inhibition of calcium ion channels (Kleuss et al. 1991). Gaz couples to almost all of the Gi-linked receptors, and yet its extreme C-terminus diverges from those of Gaj subtypes (Ho and Wong 1998). Several loop regions adjacent to both termini of Ga subunit are also important for defining the receptor coupling property of G protein. Multiple regions of Ga16 are required for interacting with C5a receptor (Lee et al. 1995) and one of them are the residues 220-240 (including the a2/p4 loop) of Ga16. However, mutations of the a2/p4 loop of Gas do not impede receptor coupling (Grishina and Berlot, 2000). Instead, the same study points to the importance of the a3/p5 loop in receptor coupling, where homologous mutations of Gas residues to those of Gaj2 increase the affinity of the mutant to P-adrenoceptor. The a4 helix and a4/p6 loop has been demonstrated to be a major receptor contact site in two successive chimera studies (Bae etal. 1997). In addition to the C-terminal tail, the a4/p6 loop can also be crosslinked with the third intracellular loop of rhodopsin, suggesting a direct contact between the two structural domains (Cai et al. 2001). Sequence alignment of all Ga subunits showed that Ga16 has particularly long a4/p6 loop, and its promiscuous receptor coupling property is greatly impaired when it is trimmed down to a length comparable to other Ga subunits (authors' unpublished results). It is obvious that different modes of actions may occur between specific receptors and G proteins.

The N-terminus of Ga is also involved in receptor recognition. Deletion of first six amino acids of Gaq can also allow the mutant to couple to Gi -linked receptors (Kostenis etal. 1997). Substitution of the N-terminal 36 residues of Gat1 with the corresponding region of Gaz enables the chimera to couple to 8-opioid receptor (Ho and Wong 2000). The portion of the N-terminal helix closer to the core of Ga is in close proximity with the C-terminal tail, as revealed in the crystal structures of various Ga subunits, and the third intracellular loop of receptor (Itoh et al. 2001).

GPy complex may also be important for determining the efficiency and specificity of receptor coupling (see Gudermann etal. 1997). Apeptide derived from a cytoplasmic portion of the a2-adrenergic receptor binds to a defined site of the Gp subunit, and the identity of Gy subunit maybe involved in specifying receptor-G protein interaction. The N-terminus of Ga subunit and the C-terminus of Gy subunit are in close proximity and both are lying on the plasma membrane-facing surface of the heterotrimer (Wall et al. 1995, Fig. 3.2c). A model for the receptor-Gßy complex predicts a contact between the receptor's C-terminal tail and the Gßy dimer (Lichtarge et al. 1996, Fig. 3.2c). Another detailed mapping of the receptor-and effector-interacting surfaces of Gßy subunits suggests that blades 1-3 contribute most of the protein-protein interactions (Ford et al. 1998).

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