Dual contacts between peptide agonist ligands and the secretin receptor directly established by photoaffinity labeling.
Structural analysis of secretin in solution has demonstrated extended helical domains within both amino- and carboxyl-terminal halves, with a possible turn in between. However, the conformation of this peptide as it resides in its binding site within the receptor has not been established. In the work reported here, we performed affinity labeling of the secretin receptor with radioiodinated secretin analogues having photolabile benzoyl-phenylalanine residues positioned in each half of the peptide. The probes had sites of covalent attachment in positions 6 and 22, and have been recently synthesized and characterized to represent high affinity agonist ligands. Both covalently labeled the secretin receptor in a saturable, specific, and efficient manner. After purification of the labeled receptor, we used a series of chemical and enzymatic cleavage techniques to define the domain of labeling. We complemented this by receptor mutagenesis, followed by additional cleavage and Edman degradation sequencing to refine our insights into the labeled residues. This has allowed us to demonstrate that sites of attachment were both within the extracellular aminoterminal domain of the receptor. Of particular interest, both probes labeled residues within the amino-terminal thirty residues at the distal end of the receptor. It will be particularly interesting to use these molecular approximations to model the binding domain of this important receptor.
Dong
M
M
Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.
Wang
Y
Y
Miller
L J
LJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012633:Secretin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor; D012633:Secretin