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.
Staley
J
J
Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC 20037.
Coy
D H
DH
Jensen
R T
RT
Moody
T W
TW
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