Characterization of the bombesin receptor on mouse pancreatic acini by chemical cross-linking.
Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18-27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of 125I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used 125I-GRP and chemical cross-linking techniques to characterize the mouse pancreatic BN receptor. After binding of 125I-GRP to membranes, and incubation with various chemical cross-linking agents, cross-linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18-27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of 125I-GRP to membranes or cross-linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure.
Huang
S C
SC
Digestive Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Yu
D H
DH
Wank
S A
SA
Gardner
J D
JD
Jensen
R T
RT
D000581:Amidohydrolases; D000681:Amylases / Q000378:metabolism; D000818:Animals; D001839:Bombesin / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D019886:Gastrin-Releasing Peptide; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008297:Male; D051379:Mice; D015287:Neurokinin B / Q000031:analogs & derivatives / Q000378:metabolism; D010179:Pancreas / Q000737:chemistry* / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000378:metabolism; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D019886:Gastrin-Releasing Peptide; C040794:neuromedin C; D015287:Neurokinin B; C038393:neuromedin B; D000681:Amylases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D001839:Bombesin