Identification of two pairs of spatially approximated residues within the carboxyl terminus of secretin and its receptor.
The carboxyl-terminal domains of secretin family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within secretin and the prototypic secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant secretin receptors (V13M and V16M) and sequence analysis. We also developed a new probe incorporating a photolabile Bpa at position 26 of secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC(50) = 72 +/- 6 pm) and bound to the secretin receptor specifically and with high affinity (K(i) = 10.3 +/- 2.4 nm). It covalently labeled the secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36). These new insights, along with our recent report of contact between residue 6 within the amino-terminal half of secretin and this same amino-terminal region of this receptor (Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.
Dong
M
M
Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry/Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Asmann
Y W
YW
Zang
M
M
Pinon
D I
DI
Miller
L J
LJ
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000378:metabolism; D012633:Secretin / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D008024:Ligands; D019920:Photoaffinity Labels; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor; D012633:Secretin; D010649:Phenylalanine; C053764:benzoylphenylalanine; D000409:Alanine; D005998:Glycine