Reconstitution of receptors and GTP-binding regulatory proteins (G Proteins) in Sf9 Cells: a direct evaluation of selectivity in receptor.G protein coupling

Barr, Alastair J. and Brass, Lawrence F. and Manning, David R. (1997) Reconstitution of receptors and GTP-binding regulatory proteins (G Proteins) in Sf9 Cells: a direct evaluation of selectivity in receptor.G protein coupling. Journal of Biological Chemistry, 272 (4). pp. 2223-2229. ISSN 0021-9258

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Official URL: http://dx.doi.org/10.1074/jbc.272.4.2223

Abstract

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. ?, ?1, and ?2 subunits). Membranes were incubated with [35S]guanosine 5?-(3-O-thio)triphosphate (GTP?S) ± an agonist, and the amount of radiolabel bound to the ? subunit was quantitated following immunoprecipitation. When expressed without receptor (but with ?1?2), the G protein subunits ?z, ?12, and ?13 did not bind appreciable levels of [35S]GTP?S, consistent with a minimal level of GDP/[35S]GTP?S exchange. In contrast, the subunits ?s and ?q bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTP?S to ?z but not to ?12, ?13, or ?s. Binding to ?z was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTP?S binding to ?z and more robust increases in binding to ?q, ?12, and ?13. Binding of [35S]GTP?S to ?s was strongly enhanced only by the activated ?1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.

Item Type: Article
Subjects: University of Westminster > Science and Technology > Life Sciences, School of (No longer in use)
Depositing User: Rachel Wheelhouse
Date Deposited: 20 Jul 2012 13:05
Last Modified: 20 Jul 2012 13:05
URI: http://westminsterresearch.wmin.ac.uk/id/eprint/10862

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