Functional Complementation Of Pairs Of Gpcrg Protein Fusions

3.1. GPCR-G Protein Fusions

The first GPCR-G protein fusion was constructed between the P2-adrenoceptor and the a-subunit of the long isoform of the G protein Gs. When this was expressed in S49 cyc- cells lacking expression of Gas, the P-adrenoceptor agonist isoprenaline was able to stimulate adenylyl cyclase (32), thus confirming the functionality of both elements of the fusion construct. Although the fusion construct had several interesting characteristics (32), it was initially viewed as little more than a curiosity. However, over the intervening period, a wide range of GPCRs and G proteins have been used to generate similar fusions (33,34). These have been used to address issues such as the selectivity of GPCRs in activating different G proteins (35), the role and regulation of posttranslational acylation in the function and cellular location of GPCRs and G proteins (36-38), as reagents to screen for agonists at "orphan" GPCRs (39,40) and, most recently, to explore the basis and selectivity of GPCR dimerization (41).

GPCR-G protein fusions can also be considered as bifunctional polypeptides because, although they are generated from a single open frame, they contain the sequence and functionality of both GPCR and G protein. Therefore, mutants that eliminate the ability of agonists to generate a signal corresponding to activation of the G protein can be produced by alterations in the sequence of either the GPCR or the G protein element. This allows for the production of distinct pairs of fusions that are individually inactive but have the potential to complement function if they interact. Two obvious regions can be targeted for the G protein segment of the fusion. All G protein a-subunits have a conserved Val-Gly-Gly-Gln-Arg sequence (Table 1), where mutation of the second Gly to Ala generates a form of the G protein that is unable to exchange guanosine diphosphate (GDP) for guanosine triphosphate (GTP) and hence become activated. This provides a simple strategy to generate GPCR-G protein fusion proteins that are unable to respond to agonist ligands even though the GPCRs linked to such mutants are wild-type and able to bind ligands. Equally, because it is well established that the extreme C-terminal region of most G protein a-subunits is a key contact domain for GPCR-mediated activation (42), judicious mutation in this region can produce forms of the G protein that are not responsive to GPCRs. For example, because pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation of the Cys residue four amino acids from the C-terminus of all widely expressed Gj-family G protein a-subunits prevents GPCR-mediated activation of these G proteins, Bahia et al. (43) replaced this residue in Gai1 with each of the other naturally occurring amino acids and then assessed the ability of each of the mutants to be activated by agonist occupancy of the a2A-adrenoceptor. Substitution of the Cys with more hydrophilic amino acids reduced coupling effectiveness, and there was no significant activation with either positively or negatively charged amino acids at this position. Similar data have been produced for activation of equivalently mutated forms of Gai3 by the 5-HT1A receptor (44). Several of the Gai1 mutations have been constructed into fusion proteins with the a2A-adrenoceptor and have been used to monitor the effects on these alterations on information transfer from GPCR to G protein, which was measured as alterations in agonist potency and relative efficacy (45,46). Similarly, mutation of the Tyr four amino acids from the C-terminal of Ga11 resulted in reduction of its ability to be activated by the a1b-adrenoceptor (47). With Asp at this position, no activation was observed by the agonist-occupied a1b-adrenoceptor, both in co-expression studies and when the modified G protein was constructed into a fusion with this GPCR (47). Although it has not been explored in such detail, it is well known that the lack of function of Gas in the S49

Table 1

A Generic Strategy to Generate Inactive G Protein a-Subunits

Table 1

A Generic Strategy to Generate Inactive G Protein a-Subunits

G a-subunit

Species

Sequence

Gs

Human

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