Abstract

Adenylyl cyclase (AC) converts ATP into cyclic AMP (cAMP), an important second messenger in cell signaling. Heterotrimeric G proteins and other regulators are important for control of AC activity. Depending on the AC isoform, Gβγ subunits can either conditionally stimulate or inhibit cAMP synthesis. We previously showed that the Gα_s-βγ heterotrimer binds to the N terminus (NT) of type 5 AC (AC5). We now show that Gβγ binds to the NT of a wide variety of AC isoforms. We hypothesized that Gβγ/AC5 interactions involving inactive heterotrimer and Gβγ stimulation of AC5 were separable events. Mutations of the Gβγ “hotspot” show that this site is necessary for AC5 stimulation but not for interactions with the first 198 aa of AC5NT, which is a G protein scaffolding site. This contrasts with AC6, where the Gβγ hotspot is required for both interactions with AC6NT and for stimulation of AC6. Additionally, the SIGK hotspot peptide disrupts Gβγ regulation of AC isoforms 1, 2, and 6, but not AC5. Gβγ also binds the C1/C2 catalytic domains of AC5 and AC6. Finally, cellular interactions with full-length AC5 depend on multiple sites on Gβγ. This suggests an isoform-specific mechanism in which bound Gβγ at the AC5NT is ideally situated for spatiotemporal control of AC5. We propose Gβγ regulation of AC involves multiple binding events, and the role of the AC NT for mechanisms of regulation by heterotrimeric G protein subunits is isoform-specific.