SOLUBILIZATION OF RECEPTORS FOR PANCREATIC-POLYPEPTIDE FROM RAT-LIVERMEMBRANES

We have previously identified, on rat liver microsomes and plasma memb ranes, proteins that bind pancreatic polypeptide (PP) with high affini ty and specificity and that may serve as receptors for a hepatic effec t of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterizatio n of these proteins requires the solubilization of receptors with cons erved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent olamidopropyl)-dimethylammoni o]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous pepti des neuropeptide Y and peptide YY). Gel filtration chromatography show ed different degrees of receptor aggregation related to different conc entrations of CHAPS in the eluent. To characterize the structure of th ese solubilized receptors, the chemical cross-linker N-(5-azido-2-nitr obenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyze d by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radi oactive band with an apparent molecular weight (M(r)) of 46,000 was de tected that was inhibited by unlabeled PP with a half-maximal inhibito ry concentration of similar to 10-(8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular we ight of PP. The migration of this complex was not affected by the redu cing agent dithiothreitol, suggesting the absence of disulfide bonding . The solubilization and identification of a bioactive hepatic PP rece ptor will allow further characterization and purification of this rece ptor and will lead to the clarification of the interaction between PP and the digestive system.