title: Identification of an interaction between residue 6 of the natural peptide ligand and a distinct residue within the amino-terminal tail of the secretin receptor.
abstract: Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding. We recently used this technique to demonstrate the proximity between a residue within the carboxyl-terminal half of a secretin-like ligand and the amino-terminal domain of the secretin receptor (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). In this work, we have developed another novel radioiodinatable secretin analogue ([Bpa6,Tyr10]rat secretin-27) that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) residue into position 6 of the amino-terminal half of the ligand and used this to identify a specific receptor residue proximate to it. This probe specifically bound to the secretin receptor with high affinity (IC50 = 13.2 +/- 2.5 nM) and was a potent stimulant of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary-SecR cells (EC50 = 720 +/- 230 pM). It covalently labeled the secretin receptor in a saturable and specific manner. Cyanogen bromide cleavage of this molecule yielded a single labeled fragment that migrated on an SDS-polyacrylamide gel at Mr = 19,000 that shifted to 10 after deglycosylation, most consistent with either of two glycosylated fragments within the amino-terminal tail. By immunoprecipitation with antibody directed to epitope tags incorporated into each of the two candidate fragments, the most distal fragment at the amino terminus was identified as the domain of labeling. The labeled domain was further refined to the first 16 residues by endoproteinase Lys-C cleavage and by cyanogen bromide cleavage of another receptor construct in which Val16 was mutated to Met. Radiochemical sequencing of photoaffinity-labeled secretin receptor fragments established that Val4 was the specific site of covalent attachment. This provides the first residue-residue contact between a secretin ligand and its receptor and will contribute substantially to the molecular understanding of this interaction.
authors.affiliation: Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry/Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
authors.forename: M
authors.initials: M
authors.lastname: Dong
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: E M
authors.initials: EM
authors.lastname: Hadac
authors.forename: D I
authors.initials: DI
authors.lastname: Pinon
authors.forename: E
authors.initials: E
authors.lastname: Holicky
authors.forename: L J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D008024:Ligands; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D012633:Secretin / Q000031:analogs & derivatives* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014633:Valine / Q000378:metabolism
chemical_list: D008024:Ligands; D019920:Photoaffinity Labels; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor; D012633:Secretin; D010649:Phenylalanine; C015567:secretin, Tyr(6)-; C053764:benzoylphenylalanine; D014633:Valine