2-AZIDO-[P-32]NAD+, A PHOTOACTIVATABLE PROBE FOR G-PROTEIN STRUCTURE - EVIDENCE FOR HOLOTRANSDUCIN OLIGOMERS IN WHICH THE ADP-RIBOSYLATED CARBOXYL TERMINUS OF ALPHA INTERACTS WITH BOTH ALPHA-SUBUNITS AND GAMMA-SUBUNITS

A radioactive and photoactivatable derivative of NAD+, 2-azido-[adenylate-32P]NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the α subunit (αT) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of αT followed by light activation of the azide moiety of 2-azido-[adenylate-32P]ADP-ribose produced four crosslinked species involving the α and γ subunits of the GT heterotrimer: an α trimer (α-α-α), an α-α-γ crosslink, an α dimer (α-α), and an α-γ crosslink. The α trimer, α-α-γ complex, α dimer, and α-γ complexes were immunoreactive with αT antibodies. The α-α-γ and the α-γ complexes were immunoreactive with antisera recognizing γ subunits. No evidence was found for crosslinking of αT to βT subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-[adenylate-32P]ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of αT in the crosslinked oligomers to α monomers, indicative of intermolecular photocrosslinking, and to γ monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the αT carboxyl terminus, is oriented toward and in close proximity to the γ subunit.