title: Interaction among four residues distributed through the secretin pharmacophore and a focused region of the secretin receptor amino terminus.
abstract: The amino terminus of the secretin receptor (SecR) is known to be critical for natural agonist action, although the role it plays is still unclear. We have demonstrated that photolabile residues within both the amino-terminal (position 6) and carboxyl-terminal (positions 22 and 26) halves of secretin each covalently label receptor amino-terminal tail residues [Dong et al., J Biol Chem, 274:19161-19167 (1999), 274:903-909 (1999), and 275:26032-26039 (2000)]. Here, we extend this series of studies with an additional probe having its site of covalent attachment in a distinct region of the peptide, between amino- and carboxyl-terminal helical domains. This probe incorporated a photolabile (epsilon-p-benzoylbenzoyl)lysine in position 18 and a site for oxidative radioiodination [(tyrosine(10),(benzoyl-benzoyl)lysine(18))rat secretin-27]. This analog represented a full agonist, stimulating cAMP accumulation in Chinese hamster ovary-SecR cells in a concentration-dependent manner. It bound to the SecR specifically and saturably, and was able to efficiently label that molecule within its amino terminus. Sequential specific cleavage, purification, and sequencing demonstrated that this probe labeled receptor residue arginine(14), in the same subdomain as that labeled by previous probes. Consistent with the importance of this residue, alanine replacement mutagenesis (R14A) resulted in substantial reductions in the potency (127-fold) and binding affinity (400-fold) of secretin relative to its action at the wild-type receptor. We have been able to accommodate all four extant pairs of residue-residue approximations between divergent regions of the secretin pharmacophore and the first forty residues of the SecR into a credible molecular model of this interaction. Additional experimentally derived constraints will be necessary to determine the spatial positioning of this complex with the remainder of the SecR.
authors.affiliation: Center for Basic Research in Digestive Diseases, Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
authors.forename: Maoqing
authors.initials: M
authors.lastname: Dong
authors.forename: Mengwei
authors.initials: M
authors.lastname: Zang
authors.forename: Delia I
authors.initials: DI
authors.lastname: Pinon
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Li
authors.forename: Terry P
authors.initials: TP
authors.lastname: Lybrand
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012633:Secretin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D011994:Recombinant Proteins; C029937:secretin receptor; D012633:Secretin