Inhibition of adenylyl cyclase by GTPase-deficient G[alpha]i is mechanistically different from that mediated by receptor-activated G[alpha]i

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize G.sub.i proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of G[alpha].sub.i by measuring the loss of activity upon homologous replacements of putative regions of constitutive active G[alpha].sub.i mutants. However, whether such findings can indeed be translated into the context of a receptor-activated G[alpha].sub.i have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of G[alpha].sub.i1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the [alpha]3/[beta]5 loop of G[alpha].sub.i1. Subsequent cAMP assays support a cooperative action of the [alpha]3/[beta]5 loop, the [alpha]4 helix, and the [alpha]4/[beta]6 loop in mediating AC inhibition by G[alpha].sub.i1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated G[alpha].sub.i1 to inhibit AC, and identified a previously unknown AC-interacting domain of G[alpha].sub.i subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.