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

We report the direct interaction of 07 with the third intracellular (13) loop of the M-r and M;j-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loops of the M2- and the Ms-MR were expressed in bacteria and purified as GST 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 13 peptide affinity matrix. However, stimulation of subimit dissociation by GT?7S/Mg2+ resulted in the retention of G;3-y, but not the Ga subunit, by the M2- and M;, MR i:J pfptide resin. Purified G3-y bound to the Ms-MR I3 peptide with an apparent affinity similar to that observed for the G3~j binding domain of the receptor kinase GRK2 and Bruton ty rosi ne kînase, whereas transducin 7 was not recognized by the M.j-MR S3 peptide. Effective phosphorylation of the Mvl-MR i3 peptide by GRK2 required both G .7 and lipid as is the case for the intact receptor. Incubation of GRK2 with the 13 peptide in the presence of G/?7 resulted in the formation of a functional ternary complex in which G3f 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 G7 to act as a docking protein may also serve to provide an interface for this class of membrane bound receptor to an expanded array of signalling pathways.