AJP - Gastrointestinal and Liver Physiology, Vol 258, Issue 5 803-G809, Copyright © 1990 by American Physiological Society
Molecular characterization of bombesin receptors on rat pancreatic acinar AR42J cells
P. Singh, E. Draviam, Y. S. Guo and A. Kurosky
Department of Surgery, University of Texas Medical Branch, Galveston 77550.
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.
