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.
Highlights
The secretin receptor is a prototypic member of the Class II family within the superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors [2]
Included among these studies is the first demonstration of the affinity labeling of a segment of the amino terminus of the secretin receptor that represents the first 30 residues using a probe with its photolabile site of attachment in position 22 in the carboxyl-terminal half of the ligand [1]
Of particular interest is the correlation between the structures of groups of ligands and the structural themes expressed within the groups of G protein-coupled receptors that they recognize [25, 26]
Summary
The secretin receptor is a prototypic member of the Class II family within the superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors [2]. Broad receptor domains of importance in ligand binding have been identified This is based predominantly on mutagenesis studies, including analysis of chimeric receptors, deletion constructs, and site mutants [5,6,7,8,9,10,11,12,13]. These studies have postulated a critical role for the amino-terminal domain of the Class II receptors in binding their natural peptide ligands. The ligand binding domain was localized by a series of targeted enzymatic and chemical fragmentation reactions, and the labeled residue was identified as Val using radiochemical Edman degradation sequencing
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