Role of G Protein-coupled Receptor Kinase 4 and ॆ-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases

Abstract

The metabotropic glutamate 1 (mGlu 1 ) 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 mGlu 1 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 mGlu 1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated G␣ q . In cells transfected with GRK4 and exposed to agonist, ␤-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu 1 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 mGlu 1 receptor internalization, reduced by 70 -80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu 1 receptor. The agonist-stimulated differential sorting of the mGlu 1 receptor and ␤-arrestin as well as the activation of MAP kinases by mGlu 1 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 mGlu 1 receptor by different mechanisms and that ␤-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.Metabotropic glutamate (mGlu) 1 receptors, which are activated by the excitatory amino acid glutamate, are part of an original family of G protein-coupled receptors (GPCR) called the family 3 GPCRs (1-3). These include all the mGlu receptor subtypes, Ca 2ϩ -sensing and GABA B receptors, and some putative olfactory, pheromone, and taste receptors. Eight subtypes of mGlu receptors have been identified, which are implicated in different aspects of physiology and pathology of the central nervous system. Group I mGlu receptors (mGlu 1 and mGlu 5 ), which stimulate polyphosphoinositide hydrolysis by coupling to G q , are localized in the peripheral parts of postsynaptic dendrites and contribute to the regulation of synaptic plasticity. For example, mGlu 1 receptor present in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Similar to many other GPCRs, the signal transduction of the mGlu 1 receptor is strictly regulated by multiple mechanisms acting at different levels of signal propagation (1). After prolonged or repeated stimulation, receptors are profoundly desensitized. Protein kinase C is clearly involved in this process, although a protein kinase C-independent component of mGlu 1 receptor desensitization was also observed (4). The activated ␣ subunit of the G q (G␣ q ) ca...

G protein-coupled receptor kinase 4γ interacts with inactive Gαs and Gα13

“…Initially weak interactions between GRK4γ and many of the Gα subunits were observed, but only the AU1-Gα 13 interaction with GRK4γ proved to be specific when using the wild-type and QL mutants. This supports previous findings that GRK4 does not interact with Gα q or Gα 11 [18]. In accordance with figure 1, GRK4γ is co-immunoprecipitated with inactive AU1-Gα 13 and to a lesser extent when treated with AlF 4 − (Fig.…”

Non-visual GRKs: are we seeing the whole picture?

“…Initial surprises came from work on the a 1B -adrenoceptor [71] and the endothelin ET A and ET B receptors [72], which demonstrated that overexpression of either wild-type GRK2 or a catalytically inactive mutant form of this protein suppressed receptorstimulated Ga q/11 -mediated phospholipase C (PLC) activation. This has now been observed with several other PLC-linked GPCRs in a variety of cell backgrounds [24,28,37,73]. Although these phosphorylationindependent actions of GRK2 were initially attributed to the ability of GRK2 to sequester Gbg-subunits [67], it now seems more likely that GRK2 selectively binds to the activated form of Ga q/11 (although not to Ga s , Ga i or Ga 12/13 ) to suppress PLC-b activation.…”

“…However, GRK4 appears to play a specific role in the regulation of GPCRs in tissues in which its expression is relatively high (e.g. cerebellar Purkinje cells [21,28] and the renal medulla [20]). Studies using antisense oligonucleotides to reduce selectively the expression of GRK2 or GRK4 in rat Purkinje cells or human renal proximal tubule cells have revealed key differences between these kinases.…”

Imaging of Muscarinic Acetylcholine Receptor Signaling in Hippocampal Neurons: Evidence for Phosphorylation-Dependent and -Independent Regulation by G-Protein-Coupled Receptor Kinases

“…Evidence for the involvement of GRK4, GRK5, and GRK6 in neuronal GPCR regulation has also been presented. Thus, De Blasi and colleagues have made a strong case for GRK4 involvement in the desensitization of type 1 metabotropic glutamate receptors in Purkinje cells (Sallese et al, 2000;Iacovelli et al, 2003), and targeted GRK5 (Gainetdinov et al, 1999) and GRK6 (Gainetdinov et al, 2003) gene knock-out by homologous recombination has provided evidence for an in-volvement of these GRKs in muscarinic acetylcholine (mACh) receptor and dopamine receptor regulation in vivo, respectively.…”