Role of G protein-coupled receptor kinase 4 and beta-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases.

The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G proteincoupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gq. In cells transfected with GRK4 and exposed to agonist, -arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of -arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70–80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and -arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of -arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing -arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1 receptor by different mechanisms and that -arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.