Influence of Differential Stability of G Protein beta gamma Dimers Containing the gamma_(11) Subunit on Functional Activity at the M_1 Muscarinic Receptor,A_1 Adenosine Receptor,and Phospholipase C-beta

摘要

G gamma_(11) is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit.To study the effect of different G beta-binding partners on ganna_(11) function,four recombinant beta_gamma dimers,beta_1gamma_2,beta_4gamma2,beta_1gamma_(11) and beta_4gamma_(11),were characterized in a receptor reconstitution assay with the G_q-linked M_1 muscarinic and the G_(il)-linked A_1 adenosine receptors.The beta_4gamma_(11) dimer was up to 30-fold less efficient than beta_4gamma_2 at promoting agonist-dependent binding of [~(35)S]GTPyS to either alpha_q,or alpha_(il).Using a competition assay to measure relative affinities of purified beta gamma dimers for alpha,the beta_4gamma_(11) dimer had a 15-fold lower affinity for G_(il) alpha than beta_4gamma_2 centre dot Chromatographic characterization of the beta_4gamma_(11) dimer revealed that the beta gamma is stable in a heterotrimeric complex with G_(il) alpha;however,upon activation of a with MgCl_2 and GTPyS under nondenaturing conditions,the beta_4 and gamma_(11) subunits dissociate.Activation of purified G_(il) alpha:beta_4gamma_(11) with Mg~(+2)/GTPyS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity;however,when this activation preceded reconstitution into vesicles,PLC-beta activity was markedly diminished.In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A_1 adenosine receptor,beta_4gamma_(11) was as effective as beta_4gamma_2 when coexpressed with G_(il) a and receptor.However,G_(il) alpha:beta_4gamma_(11)-induced high-affinity binding was up to 20-fold more sensitive to GTPyS than G_(il) alpha:beta_4gamma_2-induced high-affinity binding.These results suggest that the stability of the beta_4gamma_11 dimer can modulate G protein activity at.the receptor and effector.