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.[1]References
- Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor. Luthin, D.R., Eppler, C.M., Linden, J. J. Biol. Chem. (1993) [Pubmed]
Annotations and hyperlinks in this abstract are from individual authors of WikiGenes or automatically generated by the WikiGenes Data Mining Engine. The abstract is from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.About WikiGenesOpen Access LicencePrivacy PolicyTerms of Useapsburg
