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Activation of the GTP-binding protein Gq by rhodopsin in squid photoreceptors.

ABSTRACT: Photoaffinity labelling by a GTP analogue has been used to identify a 42 kDa band as the major G alpha subunit in squid photoreceptor membranes, recently identified by partial sequence analysis to be a member of the Gq sub-group of GTP-binding proteins [Pottinger, Ryba, Keen & Findlay (1991) Biochem. J. 279, 323-326]. Guanine-nucleotide-binding displacement analysis gave a stoichiometry of 1 G-protein per 12.5 rhodopsin molecules, the same as in vertebrate rod photoreceptors. Binding was not detected above background in the dark, but was rapidly activated by light. Unlike vertebrate transducin, this G-protein is very temperature-sensitive. GTP binding is maximal at temperatures less than 10 degrees C and is much decreased after several minutes above 18 degrees C. The light-stimulated GTPase rate is maximal around 10 degrees C, above which the loss of binding sites counteracts the increase in hydrolytic rate per site. Earlier studies described light-sensitive G alpha components of 40 and 45 kDa, by ADP-ribosylation in the presence of cholera and pertussis toxins. These are now shown to be very minor components, as the prolonged treatment at elevated temperature required for ADP-ribosylation is sufficient to inactivate the major G alpha totally. Unlike the minor G alpha components, the 42 kDa G alpha is not inhibited by Ca2+.

SUBMITTER: Nobes C

PROVIDER: S-EPMC1133198 | biostudies-other | 1992 Oct

REPOSITORIES: biostudies-other

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Light-dependent GTP-binding proteins in squid photoreceptors.

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Specific role for p85/p110beta in GTP-binding-protein-mediated activation of Akt.

Project description:We prepared CHO (Chinese hamster ovary) cells expressing both IR (insulin receptor) and A1R (A1 adenosine receptor). Treatment of the cells with insulin or PIA [N6-(2-phenylisopropyl)adenosine], a specific A(1)R agonist increased Akt activity in the cells in a PI3K- (phosphoinositide 3-kinase) dependent manner. Transfection of p110beta into the cells augmented the action of PIA with little effect on insulin. Introduction of a pH1 vector producing shRNA (short hairpin RNA) that targets p110beta abolished PIA-induced Akt activation. By contrast, an shRNA probe targeting p110alpha did not impair the effects of PIA. The effect of PIA in p110alpha-deficient cells was attenuated effectively by both Deltap85 and betaARK-CT (beta-adrenergic receptor kinase-C-terminal peptide). A Deltap85-derived protein possessing point mutations in its two SH2 domains did not impair PIA action. These results suggest that tyrosine-phosphorylated proteins and Gbetagamma (betagamma subunits of GTP-binding protein) are necessary for the specific function of p110beta in intact cells. The p110beta-middle (middle part of p110beta) may play an important role in signal reception from GPCRs (GTP-binding-protein-coupled receptor), because transfection of the middle part impaired PIA sensitivity.

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Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region.

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