Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.
The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and was purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose and ligand affinity chromatography. The purified receptor displayed a single diffuse band with a Mr of 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment of the receptor with N-glycanase, removing N-linked oligosaccharide moieties, the protein yielded a Mr = 38,000 band. These results agree with the Mr value estimated for the GRP receptor that was labeled on Swiss 3T3 cells by cross-linking to 125I-GRP1-27. GRP1-27 bound to the purified receptor with a Kd of 0.038 +/- 0.019 nM. By comparison, the soluble receptor in unfractionated extracts and intact membranes displayed a Kd for GRP1-27 of 0.036 +/- 0.003 nM and 0.13 +/- 0.04 nM, respectively. The relative potencies of a series of GRP analogs for the soluble receptor and intact membranes indicated that the extraction procedure did not significantly alter the receptor's ligand binding specificity. However coupling of the receptor to its guanyl nucleotide regulatory protein was not maintained in the soluble extract, and a G-protein did not co-purify with the receptor. Physiological concentrations of NaCl greatly inhibited the binding of some GRP analogs to the receptor, while the binding of other analogs was not affected. A domain on the GRP molecule involving Lys-13 or Arg-17 was identified which promoted binding to the GRP receptor under conditions of low ionic strength. These findings aided the development of an effective ligand affinity resin for the purification of the GRP receptor.
Feldman
R I
RI
Triton Biosciences Inc., Alameda, California 94501.
Wu
J M
JM
Jenson
J C
JC
Mann
E
E
D000818:Animals; D001667:Binding, Competitive; D001839:Bombesin / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019886:Gastrin-Releasing Peptide; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism; D014462:Ultrafiltration
D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D019886:Gastrin-Releasing Peptide; D001839:Bombesin