Multiple extracellular loop domains contribute critical determinants for agonist binding and activation of the secretin receptor

Martin H. Holtmann, Subhas Ganguli, Elizabeth M. Hadac, Vesile Dolu, Laurence J. Miller

Research output: Contribution to journalArticle

86 Scopus citations

Abstract

Distinct themes exist for ligand-binding domains of G protein-coupled receptors. The secretin receptor is prototypic of a recently described family in this superfamily which binds moderate-sized peptides possessing a diffuse pharmacophore. We recently demonstrated the importance of the N terminus and first loop of this receptor for secretin binding (Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995) J. Biol. Chem. 270:14394-14398). Here, we extend those findings to define another receptor domain important for agonist recognition and to focus on critical determinants within each of these domains. Extending the secretin-vasoactive intestinal polypeptide (VIP) chimeric receptor approach, we confirmed and refined the critical importance of the N terminus and the need to complement this with other domains of the secretin receptor. There was redundancy in the complementary determinants required, with the second extracellular loop able to compensate for the absence of the first loop. The first 10 residues of the N terminus of the secretin receptor were critical. Sequential segmental and site replacements permitted focusing on the His189-Lys190 sequence at the C terminus of the first extracellular loop, and on four residues (Phe257, Leu258, Ash260, and Thr261) in the N-terminal half of the second loop as providing critical determinants. All receptor constructs which expressed sensitive cAMP responses to secretin (EC50 <5 nM) hound this peptide with high affinity. Of note, one construct dissociated high affinity binding of secretin from its biological responsiveness, providing a clue to the conformational 'switch' that activates this receptor.

Original languageEnglish (US)
Pages (from-to)14944-14949
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number25
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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