G Protein-coupled Receptor Kinase 2 Negatively Regulates Chemokine Signaling at a Level Downstream from G Protein Subunits

Abstract

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupledreceptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells produced a significant reduction of the extracellular signal-regulated kinase (ERK) response to CCL2. This effect is independent of its role in receptor phosphorylation because the kinase-deficient mutant GRK2K220R was able to reduce this response, and ERK activation by CCR2BIX, a phosphorylation-defective receptor mutant, was also inhibited by GRK2. Constructs containing the G␣ q -binding RGS-like RH domain of GRK2 or its G␤␥-binding domain could not reproduce the inhibition, thus revealing that GRK2 acts downstream of G proteins. Interestingly, chemokine-driven mitogen-activated protein kinase kinase (MEK) stimulation is not affected in cells overexpressing GRK2 or GRK2K220R or in splenocytes from heterozygous GRK2 mice, where reduced kinase levels correlate with enhanced ERK activation by chemokines. We find GRK2 and MEK in the same multimolecular complex, thus suggesting a mechanism for GRK2 regulation of ERK activity that involves a direct or coordinate interaction with MEK. These results suggest an important role for GRK2 in the control of chemokine induction of ERK activation at the level of the MEK-ERK interface. INTRODUCTIONG protein-coupled receptor kinases (GRKs) are serine/threonine protein kinases that control the desensitization process of the G protein-coupled receptor family (Penela et al., 2003;Willets et al., 2003). GRK2 in particular is one of the best characterized GRK isoforms and has been shown to phosphorylate and desensitize the agonist-bound form of many G protein-coupled receptors (GPCRs) (Aragay et al., 1998b). Receptor phosphorylation is followed by high-affinity binding of arrestins to the receptor, which sterically inhibits further G protein activation. Although it is now clear that arrestins serve additional roles as multifunctional adaptor molecules, emerging new evidence also unveils unexpected roles for GRKs, including the phosphorylation of nonreceptor substrates, of non-GPCR receptors, and the association to different proteins such as phosphoinositide 3-kinase, GIT, or clathrin (Penela et al., 2003;Willets et al., 2003). Moreover, the GRK2/3 subfamily members contain a Cterminal G␤␥-binding region (Carman et al., 2000;Willets et al., 2003) and are able to inactivate G protein-dependent pathways in a phosphorylation-independent manner involving their avid interaction with G␣ q family members via their RGS-like RH N-terminal domain (Carman et al., 1999;Sallese et al., 2000).Chemokines bind to a variety of GPCRs, thus mediating chemotactic and proadhesion effects in leukocytes and other cell types (Rossi and Zlotnik, 2000). The CC chemokine receptor, CCR2, exclusively binds CCL2 produced by endothelial cells, smooth muscle cells, and macrophages in response to a variety of mediators. Dis...

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