2-azido-[32P]NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of α interacts with both α and γ subunits

Richard R. Vaillancourt, N. Dhanasekaran, Gary L. Johnson, Arnold E. Ruoho

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)3645-3649
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number10
DOIs
StatePublished - Jan 1 1990
Externally publishedYes

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Keywords

  • Cysteine-347
  • Photocrosslinking
  • Transducin oligomers

ASJC Scopus subject areas

  • General

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