Abstract
Purification of the gastrin-releasing peptide (GRP) or bombesin receptor has proved elusive in part due to technical difficulties. In the present studies, the problem of oxidized radioligand was avoided by the use of 125I-GRP, which was verified to be not oxidized by high performance liquid chromatography. Specific 125I-GRP binding (at 0 degrees C) to intact human small cell lung carcinoma NCI-H345 cells which had been subjected to a dilute acid wash was 6 fmol/10(6) cells. Inhibition of GRP degradation by human H345 cell membranes through the use of phenanthroline or phosphoramidon permitted the development of binding assays for the GRP receptor in detergent-solubilized crude membrane preparations. The solubilized GRP receptor exhibited saturable, high affinity (KD = 1.3 nM), temperature-dependent specific binding averaging 402 +/- 65 fmol/mg protein (mean +/- S.E. for eight separate membrane preparations with 125I-GRP concentration = 3 nM), with a Bmax = 434 fmol/mg protein using a gel filtration binding assay. That the GRP receptor had been solubilized was demonstrated by its failure to pellet when centrifuged at 100,000 x g for 60 min, its passage through a 0.22-micron filter without loss of binding activity, and its elution in the void volume of a Sephadex G-50 gel filtration column, but within the inclusion volume of a Sephacryl S-200 column (Ve/V0 = 1.1). Isolation of the GRP receptor from human H345 cell-solubilized membranes was achieved by ligand affinity chromatography. A unique 70-kDa band on silver-stained reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis was reproducibly eluted from GRP14-27 affinity columns by an acidic high salt buffer, but binding activity was denatured by these conditions. The protein nature of the GRP receptor was demonstrated by its sensitivity to proteases after isolation. In addition, two unique bands of 65 and 70 kDa were eluted from the GRP14-27 affinity column with GRP14-27 in neutral buffer, and this eluate possessed specific 125I-GRP binding with a stoichiometry of approximately 1:1. Thus, reported here is the isolation of a functional membrane-associated, saturable, high affinity GRP receptor with temperature-dependent binding from the solubilized membranes of human H345 cells.
Highlights
gastrin-releasing peptide (GRP) is a 27-amino acid peptide which branes through thuese of phenanthroline or phosphor- normally functions as a neurotransmitter,seacretagogue,and amidon permitted the development of binding assays a growth factor, and shares the identical C-terminal heptafor the GRP receptor in detergent-solubilized crude peptide sequence with bombesin, a 14-amino acid peptide membrane preparations
"'I-GRP Degradation by Human Small Cell Lung Carcinoma Human H345Cell Membranes-No specific binding was tions which agreed within 15% and are shown for a typical experi- detected in humanH345 cell-solubilized membranes prepared ment
When the human H345 cell-solubilized membrane sample was incubated with 3 nM lZ5I-GRPfor 10 min at 37 "Cand thenanalyzed on a Sephacryl S-200column, the bound radioactivity eluted at V J V 0= 1.1, binding assay as described under "Experimental Procedures" indicating that the binding moiety was included within the demonstrate the reproducibility of the assay
Summary
Vol 266, No 15, Issue of May 25, pp. 9486-9493.1991 Printed in U.S.A. Isolation of the Bombesin/Gastrin-releasingPeptide Receptor from Human Small Cell Lung Carcinoma NCI-H345 Cells*. Purification of the gastrin-releasing peptide (GRP) ent bindingfrom the solubilized membranes of human or bombesin receptor has proved elusivein partdue to H345 cells. In human small cell lung carcinoma (SCLC),' an autocrine growth system involving gastrin-releasing peptide (GRP, mammalian bombesin) has been identified in whichSCLC jected to a dilute acid wash was fmol/lOgcells. At 0 "C, total cell-associated radioactivity using intact human H345 cells remained low, and no specific binding was observed after 10 min (Fig. 1B). "'I-GRP Degradation by Human Small Cell Lung Carcinoma Human H345Cell Membranes-No specific binding was tions which agreed within 15% and are shown for a typical experi- detected in humanH345 cell-solubilized membranes prepared ment. EDTA, 1mM, soybean trypsin inhibpermitted the development of a binding assay for detergent- itor, 0.3%, aprotinin, 50 KIU/ml,or phenylmethylsulfonyl solubilized, saturable, high affinity GRP receptors from human H345 cell membrane suspensions. 4000 1 tion of its protein nature, and possible intramolecular disulfide bonds are described
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