Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors.
BACKGROUND
Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for "intrinsic" photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor.


METHODS
The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells.


RESULTS
Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([125I]Tyr10, pNO2-Phe22) rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation.


CONCLUSIONS
This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonist-binding domain.
Ulrich
C D
CD
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota.
Pinon
D I
DI
Hadac
E M
EM
Holicky
E L
EL
Chang-Miller
A
A
Gates
L K
LK
Miller
L J
LJ
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D012633:Secretin / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D018076:DNA, Complementary; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D011994:Recombinant Proteins; C029937:secretin receptor; D012633:Secretin