RECEPTOR DOCKING SITES FOR G-PROTEIN BETA-GAMMA-SUBUNITS - IMPLICATIONS FOR SIGNAL REGULATION

We report the direct interaction of G beta gamma with the third intrac ellular (i3) loop of the M-2- and M-3-muscarinic receptors (MR) and th e importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M-2- and the M-3-MR wer e expressed in bacteria and purified as glutathione S-transferase fusi on proteins for utilization as an affinity matrix and to generate subs trate for receptor subdomain phosphorylation, In its inactive heterotr imeric state stabilized by GDP, brain G-protein did not associate with the i3 peptide affinity matrix, However, stimulation of subunit disso ciation 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 trans ducin beta gamma was not recognized by the M-3-MR i3 peptide. Effectiv e phosphorylation of the M-3-MR peptide by GRK2 required both G beta g amma and lipid as is the case for the intact receptor, Incubation of p urified GRK2 with the i3 peptide in the presence of G beta gamma resul ted in the formation of a functional ternary complex in which G beta g amma served as an adapter protein, Such a complex provides a mechanism for specific spatial translocation of GRK2 within the cell positionin g the enzyme on its substrate, the activated receptor, The apparent ab ility of G beta gamma to act as a docking protein may also serve to pr ovide an interface for this class of membrane-bound receptors to an ex panded array of signaling pathways.