The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with βγ subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236–471) of ACI is sufficient to observe βγ-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G _sα (G _sα *) or Ca ²⁺ /calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by βγ subunits, the presence of both of these regions formed another βγ interaction site that was sufficient to observe G _sα *- or Ca ²⁺ /CaM-stimulated activity. Inhibition of AC activity attributable to interaction of βγ subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of βγ on various effectors. Moreover, the C1 region of ACI was sufficient to observe G _iα1 -elicited inhibition of Ca ²⁺ /CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G _iα1 , the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of G _iα1 are mediated on the C1 regions of both ACI and ACV. The effects of βγ on ACI can be mediated by interactions with the C1a region and a βγ interacting site formed by the C1b and C2 domains of this enzyme.

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