title: Influence of differential stability of G protein βγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-β.
abstract: Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS 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 A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.
authors.affiliation: Department of Pharmacology, University of Virginia Health System, Post Office Box 800735, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908, USA. wem2p@virginia.edu
authors.forename: William E
authors.initials: WE
authors.lastname: McIntire
authors.forename: Gavin
authors.initials: G
authors.lastname: MacCleery
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Murphree
authors.forename: Kristi R
authors.initials: KR
authors.lastname: Kerchner
authors.forename: Joel
authors.initials: J
authors.lastname: Linden
authors.forename: James C
authors.initials: JC
authors.lastname: Garrison
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D011232:Chemical Precipitation; D019281:Dimerization; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000302:isolation & purification; D044387:GTP-Binding Protein beta Subunits / Q000302:isolation & purification / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D054799:Phospholipase C beta; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013816:Thermodynamics; D014422:Turkeys; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D000906:Antibodies; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D020033:Protein Isoforms; D021122:Protein Subunits; D043682:Receptor, Adenosine A1; D043643:Receptor, Muscarinic M1; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11