Two interaction sites on mammalian adenylyl cyclase type I and II: modulation by calmodulin and Gβγ

Abstract

Mammalian ACs (adenylyl cyclases) are integrating effector molecules in signal transduction regulated by a plethora of molecules in either an additive, synergistic or antagonistic manner. Out of nine different isoforms, each AC subtype uses an individual set of regulators. In the present study, we have used chimaeric constructs, point mutations and peptide competition studies with ACs to show a common mechanism of multiple contact sites for the regulatory molecules Gβγ and calmodulin. Despite their chemical, structural and functional variety and different target motifs on AC, Gβγ and calmodulin share a two-site-interaction mechanism with Gαs and forskolin to modulate AC activity. Forskolin and Gαs are known to interact with both cytosolic domains of AC, from inside the catalytic cleft as well as at the periphery. An individual interaction site located at C1 of the specifically regulated AC subtype had been ascribed for both Gβγ and calmodulin. In the present study we now show for these two regulators of AC that a second isoform- and regulator-specific contact site in C2 is necessary to render enzyme activity susceptible to Gβγ or calmodulin modulation. In addition to the PFAHL motif in C1b of ACII, Gβγ contacts the KF loop in C2, whereas calmodulin requires not only the Ca2+-independent AC28 region in C1b but also a Ca2+-dependent domain in C2a of ACI containing the VLG loop to stimulate this AC isoform.

Adenylyl Cyclase 5 Regulation by GβγInvolves Isoform-Specific Use of Multiple Interaction Sites

“…However, owing to expression issues in E. coli, we have never been able to test this region directly (aa residues 195-238). Gbg interacts with multiple sites in AC2 to promote conditional stimulation, including the C1 and C2 catalytic domains (Diel et al, 2006(Diel et al, , 2008Boran et al, 2011). Therefore, we used several strategies to determine if Gbg interacted with the C1/C2 domains of AC5 and AC6.…”

“…This is analogous to AC2, the AC isoform most thoroughly mapped for Gbg binding. In addition to our observed binding of Gbg to AC2NT, three binding sites have been mapped to the C1 domain and another two sites on the C2 domain of AC2 (Weng et al, 1996;Diel et al, 2006Diel et al, , 2008Boran et al, 2011). AC5/6 show 65% homology to the site located within the C1a domain of AC2 (aa 339-360), whereas sites located within the C1b and C2 domains of AC2 are poorly conserved or not present in AC5/6 (Boran et al, 2011).…”

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…In contrast, in the case of ACs, there are relatively few studies on chimeras of full-length AC isoforms (Levin and Reed, 1995;Crossthwaite et al, 2005;Yoshimura et al, 2006;Diel et al, 2008). A major reason for this situation is the fact that the chimeras are unstable and, hence, functionally inactive (Seebacher et al, 2001).…”

“…AC1 forms a unique interface with its activator CaM compared with other mammalian CaM targets (Vorherr et al, 1993;Diel et al, 2008;Masada et al, 2012;Lübker and Seifert, 2015;Lübker et al, 2015a,b). Principally, such a unique protein-protein interaction interface could be exploited pharmacologically.…”

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain

“…The primary sequence of AmAC2t harbors several consensus motifs for PKC phosphorylation and might thus be prone to PKC modulation. However, a PFAHL motif that has been identified as one element for G b/g binding (Diel et al, 2006(Diel et al, , 2008 is absent from the C1b domain of AmAC2t. Although this result does not exclude a modulating effect of G b/g subunits on AmAC2t, other modes of regulation might be more likely.…”