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...

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.

The catenin p120 (p120 ctn ) is an armadillo repeat domain protein that binds to cadherins and has been shown to facilitate strong cell-cell adhesion. We have investigated a possible link between heterotrimeric G proteins and p120 ctn , and found that both G␣12 and G␣ 13 can completely and selectively abrogate the p120 ctninduced branching phenotype in different cell types. Consistent with these observations, the expression of G␣12 or G␣13 compensates for the reduction of Rho activity induced by p120 ctn . On the other hand, p120 ctn can be selectively coimmunoprecipitated with G␣12, and the coimmunoprecipitation was favored by activation of the G protein. A specific interaction between p120 ctn and G␣12Q231L was also observed in in vitro binding experiments. In addition, p120 ctn can be immunoprecipitated along with G␣12Q231L in L cells in absence of E-cadherin. Interestingly, the expression of G␣12Q231L increases the amount of p120 ctn associated with E-cadherin. These findings demonstrate that G␣12 and p120 ctn are binding partners, and they also suggest a role for G␣12 in regulating p120 ctn activity and its interaction with cadherins. We propose that the G␣12-p120 ctn interaction acts as a molecular switch, which regulates cadherin-mediated cell-cell adhesion.C adherins are transmembrane glycoproteins involved in calcium-dependent cell-cell adhesion (1). Contact between cadherins and the actin cytoskeleton is necessary for the stabilization of cell-cell adhesion and normal cell physiology. Catenins such as ␤-catenin and plakoglobin bind to the C terminus of the cadherin molecule (2-5) and are linked to the actin cytoskeleton via ␣-catenin (6, 7). The catenin p120 (p120 ctn ) is one of several proteins that bind to the juxtamembrane region of the cytoplasmic domain of E-cadherin (8). This region is known to be important for the function of cadherin (9, 10) and is important in the regulation of cell motility and metastatic invasion (10).The catenin p120 is a member of a diverse family of armadillo repeat domain proteins. Originally discovered as a substrate for the Src oncoprotein kinase (11), p120 ctn can be phosphorylated on both tyrosine and serine residues (11-13). p120 ctn has been shown to regulate the activity of Rho small GTPases, and thus influence cadherin-mediated cell adhesion and cell migration (14-16). Overexpression of p120 ctn in several different cell lines results in a variety of morphological effects, depending on the cell type and on the level of p120 ctn expression (14, 17). These morphological phenotypes have been associated with the ability of p120 ctn to inhibit RhoA (14,15). In Drosophila, Rho1 can interact with both ␣-catenin and p120 ctn (18). Interestingly, recent reports have suggested that p120 ctn is a critical component in the maintenance of steady-state cadherin levels (19-21). Moreover, p120 ctn promotes cell surface trafficking of cadherins via association and recruitment of kinesin (22).The G 12 subfamily of heterotrimeric G proteins, which is comprised of G␣ 12 and G␣ 13 s...

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