TY - JOUR
T1 - Multiple extracellular loop domains contribute critical determinants for agonist binding and activation of the secretin receptor
AU - Holtmann, Martin H.
AU - Ganguli, Subhas
AU - Hadac, Elizabeth M.
AU - Dolu, Vesile
AU - Miller, Laurence J.
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.271.25.14944
DO - 10.1074/jbc.271.25.14944
M3 - Article
C2 - 8663161
AN - SCOPUS:15844385219
SN - 0021-9258
VL - 271
SP - 14944
EP - 14949
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 25
ER -