Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs.
A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.
American Cyanamid Company, Agricultural Research Division, Princeton, New Jersey 08543.
C M
CM
Eppler
J R
JR
Zysk
M
M
Corbett
H M
HM
Shieh
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D037342:Pertussis Toxin; D010911:Pituitary Neoplasms; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism; D017481:Receptors, Somatostatin; D012995:Solubility; D013004:Somatostatin / Q000031:analogs & derivatives* / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D003432:Cross-Linking Reagents; D011480:Protease Inhibitors; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D013004:Somatostatin; D001710:Biotin; D008715:Methionine; D037342:Pertussis Toxin