Solubilization and purification of bombesin/gastrin releasing peptide receptors from human cell lines.
Bombesin/gastrin releasing peptide (BN/GRP) receptors were solubilized and purified from human glioblastoma (U-118) and lung carcinoid cell lines (NCI-H720). The U-118 cells, when extracted with CHAPS/cholesterol hemisuccinate (CHS), bound (125I-Tyr4)BN with high affinity (Kd = 2 nM) to a single class of sites (Bmax = 150 fmol/mg protein). Specific (125I-Tyr4)BN binding was inhibited with high affinity by BN, GRP, GRP14-27, and receptor antagonists such as (D-Phe6)BN6-13methylester(ME) and (D-Phe6)BN6-13 propylamide(PA) (IC50 = 2, 22, 3, 1 and 2 nM, respectively) but not GRP1-16 or BN1-12. The solubilized and cellular receptor bound peptides with similar affinity. The solubilized receptor was purified using (Lys0, Gly1-4, D-Ala5)BN and (Lys3, Gly4,5, D-Tyr6)BN3-13 PA affinity resins. When eluted from the affinity resins by NaCl, the receptor bound (125I-D-Tyr6)BN6-13ME with high affinity. The NCI-H720 BN/GRP receptor was purified 86,000-fold after extraction with CHAPS/CHS and purification using both affinity resins. SDS-PAGE analysis indicated that major 65 and 115 kDa proteins were purified. These data indicate that BN/GRP receptors can be solubilized from human cells and purified using affinity chromatography techniques with retention of ligand binding activity.
Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC 20037.
J
J
Staley
D H
DH
Coy
R T
RT
Jensen
T W
TW
Moody
D000595:Amino Acid Sequence; D001839:Bombesin / Q000031:analogs & derivatives / Q000378:metabolism; D001932:Brain Neoplasms / Q000737:chemistry / Q000473:pathology; D019886:Gastrin-Releasing Peptide; D005909:Glioblastoma / Q000737:chemistry / Q000473:pathology; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000473:pathology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D019886:Gastrin-Releasing Peptide; D001839:Bombesin