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 cancer 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 greater than 95% biologically active, with a specific activity of greater than 2,000 disintegrations.min-1.fmol-1. The maximum number of BBS receptors per cell were measured at 30 degrees C after 20-25 min of incubation; binding was submaximum at temperatures lower or higher than 30 degrees C. A single class of high-affinity binding sites (Kd = 1.77 +/- 0.21 nM) was identified for BBS on AR42J cells and nonspecific binding was less than 20-30% at all points. A total of 1.47 +/- 0.14 x 10(5) 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 approximately 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.
Singh
P
P
Department of Surgery, University of Texas Medical Branch, Galveston 77550.
Draviam
E
E
Guo
Y S
YS
Kurosky
A
A
D000818:Animals; D001667:Binding, Competitive; D001839:Bombesin / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*
D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D001839:Bombesin