The D₁Dopamine Receptor Is Constitutively Phosphorylated by G Protein-Coupled Receptor Kinase 4

Abstract

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D 1 receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the ␣ isoform of GRK4; the ␤, ␥, and ␦ isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4␣ is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D 1 receptor by GRK4␣ results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number-all of which are abolished in a D 1 receptor mutant containing T428V and S431A. The increase in internalized D 1 receptors induced by GRK4␣ phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D 1 receptor by GRK4␣ does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4␣. These observations shift the attenuation of D 1 receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization. Dopamine signaling in mammals is mediated by five G protein-coupled receptor (GPCR) proteins divided into two groups based upon sequence homology, G protein coupling, signaling pathways, pharmacological profiles, and desensitization kinetics (Sibley and Monsma, 1992;Missale et al., 1998 Upon agonist activation, GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response under continued agonist stimulation (Ferguson et al., 1996;Gainetdinov et al., 2004). Desensitization involves phosphorylation of the receptor by GRKs and/or second messenger-activated kinases (cAMP-dependent protein kinase or protein kinase C). Homologous desensitization of GPCRs involves only activated receptors and is primarily mediated by GRKs. GRKs are serine/threonine-directed protein kinases composed of seven isoforms divided into three families (Penela et al., 2003). GRK1 and GRK7 compose the rhodopsin kinase/visual family, are expressed exclusively in retina, and participate in desensitization of opsins in rods and cones (Somers and Klein, 1984;Hisatomi et al., 1998;Weiss et al., 1998). GRK2 (␤ARK1) and GRK3 (␤ARK2) were originally identified as regulating the ␤-adrenergic receptor and com- ABBREVIATIONS: GPCR, G protein-coupled receptor; ␤ARK, ␤-adrenergic ...

Ethanol Regulation of D1 Dopamine Receptor Signaling is Mediated by Protein Kinase C in an Isozyme-Specific Manner

“…The best-characterized family of kinases in this regard are the GRKs, which primarily mediate agonist-induced phosphorylation and desensitization of the D 1 receptor (Tiberi et al, 1996;Rankin et al, 2006). On the basis of the in situ phosphorylation results ( Figure 2); however, the EtOHdependent decrease of D 1 receptor phosphorylation was not changed in cells treated with DA, suggesting that the effects of EtOH are agonist-independent and may not involve GRKs.…”

“…Importantly, HEK293T cells provide a suitable cellular environment to examine D 1 receptor signaling, desensitization, and trafficking (Rankin et al, 2006). Treatment of cells with EtOH (100 mM) for 15 min significantly potentiated maximal DA-stimulated cAMP levels by B30%, without altering agonist potency ( Figure 1a).…”

“…If EtOH inhibits GRK activity, then agonist-induced desensitization should be reduced. Our previous observations indicated that GRK2 and GRK3 primarily mediate agonistinduced desensitization of the D 1 receptor (Rankin et al, 2006). Cells coexpressing the D 1 receptor and GRK2 or GRK3 were pretreated with DA to promote agonist-induced desensitization in the absence or presence of EtOH (Figure 4).…”

“…To more definitely determine the mechanism responsible for EtOH-dependent modulation of D 1 receptor signaling, we examined the effect of EtOH on D 1 receptor phosphorylation, as this post-translational modification is known to regulate D 1 receptor function primarily in a negative manner (Tiberi et al, 1996;Jiang and Sibley, 1999;Gardner et al, 2001;Mason et al, 2002;Kim et al, 2004;Rankin et al, 2006). Cells expressing the D 1 receptor were metabolically labeled with H 3 [ 32 P]PO 2 and either pretreated with EtOH for 10 min or never exposed to EtOH (control cells), followed by a 10-min treatment with either media, EtOH, DA, or DA + EtOH.…”

“…These assays were performed as described previously (Rankin et al, 2006). Briefly, transfected HEK293T cells were seeded into six-well plates coated with poly-D-lysine.…”