Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells

A biologically active, chemically defined, radioactive ligand was used for characterizing bombesin (BBS) receptors on rat pancreatic acinar cells (AR42J). [Tyr⁴]BBS, iodinated with enzymobeads and fractionated by high-performance liquid chromatography, was monitored for biological activity as evidenced by gastrin release from perfused isolated rat stomach. The monoiodinated peptide peak was >95% biologically active, with a specific activity of >2,000 disintegrations·min^-1·fmol^-1. The maximum number of BBS receptors per cell were measured at 30°C after 20-25 min of incubation; binding was submaximum at temperatures lower or higher than 30°C. A single class of high-affinity binding sites (K(d) = 1.77 ± 0.21 nM) was identified for BBS on AR42J cells and nonspecific binding was <20-30% at all points. A total of 1.47 ± 0.14 x 10⁵ specific BBS binding sites per cell were measured that were specific for BBS and gastrin-releasing peptide (GRP) analogues. Iodinated GRP-(1-27) was cross-linked to BBS receptors on AR42J cells using several bifunctional cross-linking reagents followed by polyacrylamide gel electrophoresis of the solubilized receptor complex under reducing and nonreducing conditions. A densitometric analysis of the autoradiographs demonstrated the presence of an ~80- to 85-kDa molecular form of the receptor as a major component under both reducing and nonreducing conditions. These results indicated that the receptor molecule is a single subunit without multiple chains covalently attached by disulfide bonding.