Receptor docking sites for G-protein betagamma subunits. Implications for signal regulation.
We report the direct interaction of Gbetagamma with the third intracellular (i3) loop of the M2- and M3-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M2- and the M3-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 GTPgammaS/Mg2+ resulted in the retention of Gbetagamma, but not the Galpha subunit, by the M2- and M3-MR i3 peptide resin. Purified Gbetagamma bound to the M3-MR i3 peptide with an apparent affinity similar to that observed for the Gbetagamma binding domain of the receptor kinase GRK2 and Bruton tyrosine kinase, whereas transducin betagamma was not recognized by the M3-MR i3 peptide. Effective phosphorylation of the M3-MR peptide by GRK2 required both Gbetagamma and lipid as is the case for the intact receptor. Incubation of purified GRK2 with the i3 peptide in the presence of Gbetagamma resulted in the formation of a functional ternary complex in which Gbetagamma 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 Gbetagamma 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.
Wu
G
G
Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Benovic
J L
JL
Hildebrandt
J D
JD
Lanier
S M
SM
D000818:Animals; D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000378:metabolism*; D046911:Macromolecular Substances; D010766:Phosphorylation; D011485:Protein Binding; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D018411:Spodoptera
D046911:Macromolecular Substances; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins