Secretin occupies a single protomer of the homodimeric secretin receptor complex: Insights from photoaffinity labeling studies using dual sites of covalent attachment

Maoqing Dong, Polo C.H. Lam, Delia I. Pinon, Andrew Orry, Patrick M. Sexton, Ruben Abagyan, Laurence J. Miller

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

The secretin receptor, a prototypic family B G protein-coupled receptor, forms a constitutive homodimeric complex that is stable even in the presence of hormone. Recently, a model of this agonist-bound receptor was built based on high resolution structures reported for amino-terminal domains of other family members. Although this model provided the best solution for all extant data, including 10 photoaffinity labeling constraints, a new such constraint now obtained with a position 16 photolabile probe was inconsistent with this model. As the secretin receptor forms constitutive homodimers, we explored whether secretin might dock across both protomers of the complex, an observation that could also contribute to the negative cooperativity observed. To directly explore this, we prepared six secretin analogue probes that simultaneously incorporated two photolabile benzoylphenylalanines as sites of covalent attachment, in positions known to label distinct receptor subdomains. Each bifunctional probe was a full agonist that labeled the receptor specifically and saturably, with electrophoretic migration consistent with labeling a single protomer of the homodimeric secretin receptor. No band representing radiolabeled receptor dimer was observed with any bifunctional probe. The labeled monomeric receptor bands were cleaved with cyanogen bromide to demonstrate that both of the photolabile benzoylphenylalanines within a single probe had established covalent adducts with a single receptor in the complex. These data are consistent with a model of secretin occupying a single secretin receptor protomer within the homodimeric receptor complex. A new molecular model accommodating all constraints is now proposed.

Original languageEnglish (US)
Pages (from-to)9919-9931
Number of pages13
JournalJournal of Biological Chemistry
Volume285
Issue number13
DOIs
StatePublished - Mar 26 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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