Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

Abstract

A BSTR ACTMonocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium f lux responses. This desensitization correlates with the Ser͞Thr phosphorylation of the receptor and with the transient translocation of the G proteincoupled receptor kinase 2 (GRK2, also called ␤-adrenergic kinase 1 or ␤ARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein ␤-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium f lux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant ␤ARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

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

“…This point was further addressed by analyzing the effect of GRK2 on the activation of ERK by a mutated form of the receptor, CCR2BIX (Franci et al, 1996). This receptor has serine/ threonine residues in the C-terminal tail of the protein mutated to alanines, is poorly phosphorylated by GRK2, and consequently, overexpression of GRK2 does not block CCR2BIX-induced calcium response (Aragay et al, 1998a). In cells transfected with CCR2BIX, CCL2 triggered a marked increase of ERK phosphorylation ( Figure 2B) that was potently impaired by GRK2 (70% inhibition).…”

“…In previous studies, we have analyzed the pathway of activation of MAPKs by CCL2 in monocytes and HEK293-CCR2B cells, which involves heterotrimeric G proteins, protein kinase C, phosphoinositide 3-kinase, and Ras (Aragay et al, 1998a;Jiménez-Sainz et al, 2003). We have also reported that activation of CCR2B by CCL2 leads to its rapid phosphorylation by GRK2.…”

“…GRK2 has been shown to phosphorylate the CCR2B receptor and desensitize CCL2-induced calcium signaling (Aragay et al, 1998a). To analyze whether the reduction observed in CCL2-mediated ERK activation in the presence of excess GRK2 involved receptor phosphorylation, similar experiments were performed using a kinase-defective mutant, GRK2K220R (Kong et al, 1994).…”

Phosphorylation-independent Association of CXCR2 with the Protein Phosphatase 2A Core Enzyme

“…Phosphorylation of the receptors in response to agonist stimulation appears to be a key step in termination of the receptor/G protein coupling, which results in desensitization of the receptors (4,7,8). Receptor phosphorylation may also play a role in facilitating certain chemokine receptor internalization (4).…”

“…Like other members of the GPCR superfamily, the functional status of many chemokine receptors is determined largely by the phosphorylation state (4 -6). Agonist treatment enhances phosphorylation of the receptors by protein kinases, presumably G protein-coupled receptor kinases and protein kinase C, which results in desensitization of the receptors (4,7,8). This phenomenon is common to many hormonal and neurotransmitter signaling systems (9), but the underlying mechanisms are still only partially understood, especially in the case of chemokine receptors.…”