Direct photoaffinity labeling of individual cytosolic domains of adenylyl cyclase by [32P]2'-deoxy-3'-AMP and [alpha-32P]5'-ATP

Abstract

The susceptibility of purines to form a covalent attachment with proteins upon exposure to UV irradiation was applied to adenylyl cyclase by use of [32P]2'-d-3'-AMP, a dead-end inhibitor that binds to the post-transition configuration of the enzyme. [32P]2'-d-3'-AMP was synthesized enzymatically. It and [alpha-32P]5'-ATP were used for direct photocross-linking to individually expressed cytosolic domains of adenylyl cyclase. Both the C1 domain of the type V isozyme (VC1) and the C2 domain of the type II isozyme (IIC2) were labeled, whether alone or combined, upon photolysis of [32P]2'-d-3'-AMP in the presence of acetone. Labeling of VC1 and IIC2 was greatly enhanced in the presence of PPi, was almost completely suppressed by 50 microM 2',5'-dideoxy-3'-ATP, the most potent reported P-site inhibitor of adenylyl cyclases, but was partially suppressed by 1 mM 3'-IMP, a ligand that does not inhibit the enzyme via the P-site. Neither 3':5'-cAMP nor 5'-ATP had a major effect on labeling by [32P]2'-d-3'-AMP. Direct cross-linking of VC1 with [alpha-32P]5'-ATP was substantially suppressed by 2', 5'-dideoxy-3'-ATP and partially suppressed by 2'-d-3'-AMP, whereas cross-linking of IIC2 was less affected by the 3'-triphosphate. The data imply that either cytosolic domain can interact directly with either substrate or P-site ligand and that subunit interaction modifies the susceptibility of each domain to UV-induced covalent modification by either [alpha-32P]5'-ATP or [32P]2'-d-3'-AMP.