Interactions of Muscarinic Receptors with the Heterotrimeric G Proteins Gq and G12: Transduction of Proliferative Signals

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Abstract
The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild‐type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus‐forming assay and a novel assay called R‐SAT (Receptor Selection and Amplification Technology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase‐deficient mutants (⋆) of Gαq and Gα12, wild‐type Gαq, and m5 were active in R‐SAT. Gα12⋆ and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of Gαq⋆. Synergistic effects of receptor/G protein combinations were not observed in focus‐forming assays but were readily detected by R‐SAT. Coexpression of Gαq with m5 induced constitutive activity in R‐SAT and increased the potency of agonists at m5 by 90‐fold. Gαq also evoked agonist‐dependent responses from m2 but not constitutive activity. Agonist potency was increased 10‐fold at m2 and decreased 15‐fold at m5 when these receptors were coexpressed with Gαqi5, a chimeric G protein containing the five C‐terminal residues of Gαi2, compared with coexpression with Gαq. Both Gαq and Gαqi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of Gα12 or Gα12i5 had no effect on the concentration‐response relationships of m5, but both elicited weak responses from m2. We conclude that although Gα12 is a more potent oncogene, Gαq transduces m5‐driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor‐initiated signal can be regulated at the level of G proteins in vivo.

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