N Terminus of Type 5 Adenylyl Cyclase Scaffolds Gs Heterotrimer

According to accepted doctrine, agonist-bound G protein-coupled receptors catalyze the exchange of GDP for GTP and facilitate the dissociation of G alpha and G beta gamma, which in turn regulate their respective effectors. More recently, the existence of preformed signaling complexes, which may include receptors, heterotrimeric G proteins, and/or effectors, is gaining acceptance. We show herein the existence of a preformed complex of inactive heterotrimer (G alpha(s).beta gamma) and the effector type 5 adenylyl cyclase (AC5), localized by the N terminus of AC5. GST fusions of AC5 N terminus (5NT) bind to purified G protein subunits (GDP-G alpha(s) and G beta gamma) with apparent affinities of 270 +/- 21 and 190 +/- 7 nM, respectively. GDP-bound G alpha(s) and G beta gamma did not compete, but rather facilitated their interaction with 5NT, consistent with the isolation of a ternary complex (5NT, G alpha(s), and G beta gamma) by gel filtration. The AC5/G beta gamma interaction was also demonstrated by immunoprecipitation and fluorescence resonance energy transfer (FRET) and the binding site of heterotrimer G alpha(s).beta gamma mapped to amino acids 60 to 129 of 5NT. Deletion of this region in full-length AC5 resulted in significant reduction of FRET between G beta gamma and AC. 5NT also interacts with the catalytic core of AC, mainly via the C1 domain, to enhance G alpha(s)-and forskolin-stimulated activity of C1/C2 domains. The N terminus also serves to constrain G alpha(i)-mediated inhibition of AC5, which is relieved in the presence of G beta gamma. These results reveal that 5NT plays a key regulatory role by interacting with the catalytic core and scaffolding inactive heterotrimeric G proteins, forming a preassembled complex that is potentially braced for GPCR activation.