Receptor docking sites for G-protein βγ subunits -: Implications for signal regulation

We report the direct interaction of G beta gamma with the third intracellular (i3) loop of the M-2- and M-3-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M-2- and the M-3-MR were expressed in bacteria and purified as glutathione S-transferase fusion proteins for utilization as an affinity matrix and to generate substrate for receptor subdomain phosphorylation, In its inactive heterotrimeric state stabilized by GDP, brain G-protein did not associate with the i3 peptide affinity matrix, However, stimulation of subunit dissociation by GTP gamma S/Mg2+ resulted in the retention of G beta gamma, but not the G alpha subunit, by the M-2- and M-3-MR i3 peptide resin, Purified G beta gamma bound to the M-3-MR i3 peptide with an apparent affinity similar to that observed for the G beta gamma binding domain of the receptor kinase GRK2 and Bruton tyrosine kinase, whereas transducin beta gamma was not recognized by the M-3-MR i3 peptide. Effective phosphorylation of the M-3-MR peptide by GRK2 required both G beta gamma and lipid as is the case for the intact receptor, Incubation of purified GRK2 with the i3 peptide in the presence of G beta gamma resulted in the formation of a functional ternary complex in which G beta gamma served as an adapter protein, Such a complex provides a mechanism for specific spatial translocation of GRK2 within the cell positioning the enzyme on its substrate, the activated receptor, The apparent ability of G beta gamma to act as a docking protein may also serve to provide an interface for this class of membrane-bound receptors to an expanded array of signaling pathways.