Regions on adenylyl cyclase that are necessary for inhibition of activity by beta gamma and G(ialpha) subunits of heterotrimeric G proteins

Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9551-6. doi: 10.1073/pnas.96.17.9551.

Abstract

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 beta gamma 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 beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)*) or Ca(2+)/calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves 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 G(salpha)*- 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 C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), 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(ialpha1) are mediated on the C1 regions of both ACI and ACV. The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylyl Cyclases / metabolism*
  • Calmodulin / metabolism
  • Escherichia coli
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • GTP-Binding Protein beta Subunits*
  • GTP-Binding Protein gamma Subunits*
  • GTP-Binding Proteins / metabolism*
  • Heterotrimeric GTP-Binding Proteins*
  • Protein Conformation
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship

Substances

  • Calmodulin
  • G-protein Beta gamma
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Recombinant Proteins
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • Heterotrimeric GTP-Binding Proteins
  • Adenylyl Cyclases