Functional importance of a structurally distinct homodimeric complex of the family B G protein-coupled secretin receptor

Fan Gao, Kaleeckal G. Harikumar, Maoqing Dong, Polo C.H. Lam, Patrick M. Sexton, Arthur Christopoulos, Andrew Bordner, Ruben Abagyan, Laurence J. Miller

Research output: Contribution to journalArticle

43 Scopus citations

Abstract

Oligomerization of G protein-coupled receptors has been described, but its structural basis and functional importance have been inconsistent. Here, we demonstrate that the agonist occupied wild-type secretin receptor is predominantly in a guanine nucleotide-sensitive high-affinity state and exhibits negative cooperativity, whereas the monomeric receptor is primarily in a guanine nucleotide-insensitive lower affinity state. We previously demonstrated constitutive homodimerization of this receptor through the lipid-exposed face of transmembrane (TM) IV. We now use cysteine-scanning mutagenesis of 14 TM IV residues, bioluminescence resonance energy transfer (BRET), and functional analysis to map spatial approximations and functional importance of specific residues in this complex. All, except for three helix-facing mutants, trafficked to the cell surface, where secretin was shown to bind and elicit cAMP production. Cells expressing complementary-tagged receptors were treated with cuprous phenanthroline to establish disulfide bonds between spatially approximated cysteines. BRET was measured as an indication of receptor oligomerization and was repeated after competitive disruption of oligomers with TM IV peptide to distinguish covalent from noncovalent associations. Although all constructs generated a significant BRET signal, this was disrupted by peptide in all except for single-site mutants replacing five residues with cysteine. Of these, covalent stabilization of receptor homodimers through positions of Gly243, Ile247, and Ala250 resulted in a GTP-sensitive high-affinity state of the receptor, whereas the same procedure with Ala246 and Phe240 mutants resulted in a GTP-insensitive lower affinity state. We propose the existence of a functionally important, structurally specific high-affinity dimeric state of the secretin receptor, which may be typical of family B G protein-coupled receptors.

Original languageEnglish (US)
Pages (from-to)264-274
Number of pages11
JournalMolecular pharmacology
Volume76
Issue number2
DOIs
StatePublished - Aug 1 2009

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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    Gao, F., Harikumar, K. G., Dong, M., Lam, P. C. H., Sexton, P. M., Christopoulos, A., Bordner, A., Abagyan, R., & Miller, L. J. (2009). Functional importance of a structurally distinct homodimeric complex of the family B G protein-coupled secretin receptor. Molecular pharmacology, 76(2), 264-274. https://doi.org/10.1124/mol.109.055756