Receptors for pancreastatin in rat liver membranes: molecular identification and characterization by covalent cross-linking.
High affinity binding sites for pancreastatin were identified for the first time, and their molecular characterization was performed with rat liver membranes. Using rat 125I-pancreastatin, we have studied the interaction of pancreastatin with liver membranes. Cross-linking of the tracer to the membranes was performed using the bifunctional reagent dithiobis(succinimidyl propionate). Analysis of binding under equilibrium conditions indicated the existence of one class of binding sites, with a Bmax of 15 fmol/mg of protein and an apparent Kd of 0.2 nM. The cross-linking of 125I-pancreastatin to liver membranes revealed a single band of M(r) 40,000, corresponding to the 125I-pancreastatin-receptor complex. The labeling of this complex was inhibited in the presence of rat pancreastatin (10(-10) to 10(-7) M) and in the presence of guanyl-5'-ylimidodiphosphate (10(-7) to 10(-4) M). Pretreatment of rat liver membranes with pertussis toxin did not affect pancreastatin binding or the inhibition by guanyl-5'-ylimidodiphosphate of pancreastatin binding. The specificity of pancreastatin binding was further assessed by displacement experiments with pancreastatin from other species and vasopressin. The binding of the pancreastatin-receptor complexes to Sepharose coupled to different lectins showed the glycoprotein nature of the pancreastatin receptor. These results strongly suggest that rat liver possesses a specific pancreastatin receptor, a glycoprotein of M(r) 35,000 that is coupled to a pertussis toxin-insensitive G protein in the plasma membrane.
Sánchez-Margalet
V
V
Department of Medical Biochemistry and Molecular Biology, Hospital Virgen Macarena, Medical School, University of Sevilla, Spain.
Valle
M
M
Goberna
R
R
D000818:Animals; D001665:Binding Sites; D053379:Chromogranin A; D003432:Cross-Linking Reagents; D006801:Humans; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism*; D008297:Male; D010187:Pancreatic Hormones / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*
D053379:Chromogranin A; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010187:Pancreatic Hormones; D011964:Receptors, Gastrointestinal Hormone; C029438:receptors, pancreatic polypeptide-specific; C050836:pancreastatin