Regions on adenylyl cyclase that are necessary for inhibition of astirity by beta gamma, and Gia subunits of heterotrimeric G proteins

摘要

The two large cytoplasmic domains (CI 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 CI or its Cla subdomain and CZ regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts withbeta gamma subunits of heterotrimeric G proteins. In addition, we also used a chimeric CI domain (VCIaIClb) in which the CIa region was derived from ACV and the Clb region was from ACI. By mixing the CI or Cla or VClaICIb domains with CZ regions of ACI or ACV, we have shown that the Cla region (amiuo acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active Gsa(Gsa*) or Caz+/ calmodulin (CaM). Although the Clb region and C2 domain of ACI were by thmselves not sufFicient for inhibition of activity by beta gamma, subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe Gsa* or Ca~2+ /CaM-stimulated activity. Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors. Moreover, the CI region of ACI was snfficient to observe Gial-elicited inhibition of Ca~2+/CaM-stimulated ac- tivity. Although the Cla region of ACV was sufficient for inhibition of activity by Gial, the presence of Clb region from eithcr ACI or ACV i