G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

Abstract

We investigated the regulatory effects of GRK2 on D 2 dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D 2 receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D 2 receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D 2 receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the G␤␥ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation. Dopamine receptors (DARs)3 belong to the seven-transmembrane-spanning domain GPCR family and are encoded by five distinct genes (1, 2). The five DARs consist of two subfamilies, which are defined by their structural, pharmacological, and transductional properties (3). The "D 1 -like" receptors consist of the D 1 and D 5 DARs, which activate the G proteins G S or G OLF to stimulate adenylate cyclase activity. The D 2 -like receptors include the D 2 , D 3 , and D 4 DARs and couple to G i/o proteins to inhibit adenylate cyclase and to also modulate voltagegated K ϩ or Ca 2ϩ channels. All five DAR subtypes are known to regulate critical functions within the central nervous system, including movement, learning and memory, reward and addiction, and cognition. More importantly, many of the DARs are central in the therapy of a number of neuropsychiatric disorders. Indeed, the D 2 receptor is one of the most highly validated drug targets in neurology and psychiatry. For instance, most antiparkinsonian drugs work by stimulating the D 2 DAR (4), whereas all clinically used antipsychotics are antagonists of this receptor (5, 6). As such, more knowledge concerning the regulation of the D 2 DAR might be helpful in devising new treatment strategies for D 2 DAR-related diseases.Most cells maintain homeostatic control of their responsiveness through regulatin...

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor

“…Knockdown of either GRK2 or arrestin3 significantly reduces the amount of agonist-induced D 2 receptor internalization (10,38), and overexpression of either protein enhances the amount of internalization seen in response to agonist (8,20,39,40). Furthermore, this response is potentiated when GRK2 and arrestin3 are overexpressed together (8,20,39).…”

Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor

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Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

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