Demonstration of a Direct Interaction between Residue 22 in the Carboxyl-terminal Half of Secretin and the Amino-terminal Tail of the Secretin Receptor Using Photoaffinity Labeling

Abstract

]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC 50 ‫؍‬ 68 ؎ 22 pM analogue and 95 ؎ 25 pM secretin). It bound specifically and with high affinity (K i ‫؍‬ 5.0 ؎ 1.1 nM) and covalently labeled the M r ‫؍‬ 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent M r ‫؍‬ 19,000 that shifted to M r ‫؍‬ 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.The secretin receptor is prototypic of a recently recognized family (Class II) of guanine nucleotide-binding protein (G protein) 1 -coupled receptors (1). Members of this family are believed to have the seven-transmembrane segment topology typical of the superfamily, but they share Ͻ12% homology with the extensively studied Class I receptors in the rhodopsin/␤-adrenergic receptor family, and they lack the signature sequences of this family (2, 3). Secretin family receptors have long aminoterminal domains incorporating six highly conserved Cys residues, believed to contribute to disulfide bonds that help define the family (3, 4). Indeed, this complex domain has been suggested to play a key role in agonist binding, as suggested by receptor mutagenesis studies (5-9). Other extracellular loop domains have also been implicated in complementary roles for agonist binding and receptor activation (4,5,7,10). Natural ligands for this family of receptors are all peptides longer than 27 residues, with structure-activity series suggesting the presence of diffuse pharmacophoric domains (3). Although this large diffuse pharmacophore nicely complements the multiple domains predicted to be outside the membrane bilayer, there is no working model to predict how the two molecules might interact.In this work, we attempt to establish an initial constraint that will contribute to the development of a model for the interaction of secretin with its receptor. We do this through photoaffinity labeling. This has the theoretical advantage of directly probing the domain adjacent to the photolabile residue within the probe after it binds to the receptor. Using this approach, we have successfully identified two binding contacts between photolabile analogues of cholecystokinin and its receptor (11,12).In this work, we have developed an analogue of secretin that incorporates a site for radioiodination an...

Identification of Two Pairs of Spatially Approximated Residues within the Carboxyl Terminus of Secretin and Its Receptor

“…These receptor constructs have previously been fully validated as binding secretin like wild type secretin receptor (1). Identifications of the labeled receptor fragments were further confirmed by modifications of electrophoretic migration of bands after additional mutagenesis to incorporate new specific sites of cleavage or by use of multiple independent cleavage methods.…”

“…Each of these secretin receptor constructs has been demonstrated to be expressed on the cell surface and to bind with appropriate specificity and high affinity (1,2,16). Development of new secretin receptor mutants that incorporated additional sites for CNBr cleavage in key positions was necessary for the current work.…”

“…Receptor-bearing Cell Lines-Chinese hamster ovary (CHO) cell lines stably expressing wild type secretin receptor (CHO-SecR), HA-tagged receptor constructs (CHO-SecR-HA37 and CHO-SecR-HA79), and Val 16 to Met (V16M) mutant receptors (CHO-SecR-V16M-HA37) have been established and reported previously (1,2,16). Each of these secretin receptor constructs has been demonstrated to be expressed on the cell surface and to bind with appropriate specificity and high affinity (1,2,16).…”

“…CNBr and endoproteinase Lys-C were used separately and in sequence to cleave the labeled secretin receptor and its fragments following procedures previously described (1). Affinity labeled secretin receptor was typically resolved on 10% SDS-polyacrylamide gels, and radiolabeled products of digestion were resolved on 10% NuPAGE gels using MES running buffer (Invitrogen, Carlsbad, CA).…”

“…We recently used affinity labeling to demonstrate spatial approximation between a photolabile residue within the carboxyl-terminal half of secretin (position 22) and a 30-residue segment at the distal amino terminus of its prototypic secretin receptor (1). Of interest, the amino-terminal tail of the class II G protein-coupled receptor family has remarkable structural features and major functional importance (3,4).…”