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.
Subject(s)
GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Isoenzymes/metabolism , Protein Subunits/metabolism , Receptor, Adenosine A1/metabolism , Receptor, Muscarinic M1/metabolism , Type C Phospholipases/metabolism , Animals , Antibodies/immunology , Binding Sites , Chemical Precipitation , Dimerization , GTP-Binding Protein alpha Subunits, Gq-G11/isolation & purification , GTP-Binding Protein beta Subunits/isolation & purification , GTP-Binding Protein gamma Subunits/isolation & purification , Phospholipase C beta , Protein Binding , Protein Isoforms/metabolism , Recombinant Proteins/metabolism , Spodoptera , Thermodynamics , TurkeysABSTRACT
The methods outlined in this article describe experiments that can probe the first steps in receptor:G protein interaction using defined, recombinant receptors and G proteins. The protocols have the advantages that the receptors are inserted properly in a cell membrane and that the investigator has complete control of the proteins reconstituted with the receptor. Specific mutations in the receptors or G proteins are studied easily and the protocols allow precise examination of the stoichiometry of the receptor:alpha:beta gamma interaction.
