TY - JOUR T1 - N Terminus of Type 5 Adenylyl Cyclase Scaffolds G<sub>s</sub> Heterotrimer JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1256 LP - 1264 DO - 10.1124/mol.109.058370 VL - 76 IS - 6 AU - Rachna Sadana AU - Nathan Dascal AU - Carmen W. Dessauer Y1 - 2009/12/01 UR - http://molpharm.aspetjournals.org/content/76/6/1256.abstract N2 - According to accepted doctrine, agonist-bound G protein-coupled receptors catalyze the exchange of GDP for GTP and facilitate the dissociation of Gα and Gβγ, 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 (Gαs·βγ) 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-Gαs and Gβγ) with apparent affinities of 270 ± 21 and 190 ± 7 nM, respectively. GDP-bound Gαs and Gβγ did not compete, but rather facilitated their interaction with 5NT, consistent with the isolation of a ternary complex (5NT, Gαs, and Gβγ) by gel filtration. The AC5/Gβγ interaction was also demonstrated by immunoprecipitation and fluorescence resonance energy transfer (FRET) and the binding site of heterotrimer Gαs·βγ mapped to amino acids 60 to 129 of 5NT. Deletion of this region in full-length AC5 resulted in significant reduction of FRET between Gβγ and AC. 5NT also interacts with the catalytic core of AC, mainly via the C1 domain, to enhance Gαs- and forskolin-stimulated activity of C1/C2 domains. The N terminus also serves to constrain Gαi-mediated inhibition of AC5, which is relieved in the presence of Gβγ. 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.© 2009 The American Society for Pharmacology and Experimental Therapeutics ER -