Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor.
Somatostatin (SRIF) receptors of GH4C1 cells occupied with biotinyl-NH-[Leu8,D-Trp22,Tyr25] somatostatin28 (bio-S28) have been affinity purified over streptavidin affinity columns (Eppler, C. M., Zysk, J. R., Corbett, M., and Shieh, H.-M. (1992) J. Biol. Chem. 267, 15603-15612). This procedure results in the copurification of a single subtype of SRIF receptor (SSTR2) and associated guanine nucleotide-binding proteins (G proteins) that are coupled to these receptors. For accurate quantification it was necessary to: (i) use homogenous recombinant standards; (ii) accurately assess the purity of standards; (iii) determine recovery of G proteins during sample preparation and Western blotting; and (iv) account for cross-reactivity among antisera. Four pertussis toxin-sensitive G proteins were quantified with previously characterized polyclonal antisera. Gi alpha 1 also was measured with a novel, more sensitive monoclonal antibody (7H7). Go alpha and Gi alpha 2 but not Gi alpha 1 and Gi alpha 3 were detected in membrane extracts prepared from GH4C1 cells. In contrast, the G proteins copurified with SSTR2 receptors were predominantly Gi alpha 2 (50% of total G protein) and Gi alpha 3 (36% of total G protein), whereas Go alpha and Gi alpha 1 were negligible. G beta subunits also were detected. Silver staining confirmed the absence of a 39-kDa protein, corresponding to the M(r) of Go alpha associated with purified SRIF receptor-G protein complexes. These data suggest that SRIF receptors selectively couple to two G proteins, one of which is sparsely expressed in GH4C1 cells; the data conform to the notion that SRIF receptors discriminate between similar pertussis toxin-sensitive G proteins.
Luthin
D R
DR
Department of Physiology, University of Virginia, Charlottesville 22908.
Eppler
C M
CM
Linden
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007106:Immune Sera; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D051381:Rats; D017481:Receptors, Somatostatin / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D007106:Immune Sera; D010455:Peptides; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; D019809:Streptavidin; D019204:GTP-Binding Proteins