Defective lymphocyte chemotaxis in β-arrestin2- and GRK6-deficient mice

Abstract

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated (''desensitized'') by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and ␤-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various ␤-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of ␤-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3؉ T and B220؉ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the ␤-arrestin2-and GRK6-deficient animals. Surprisingly, however, both T and B cells from ␤-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that ␤-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.L eukocytes migrate to sites of inflammation by recognizing a gradient of chemoattractants, and moving toward the chemoattractant source. This process of ligand recognition, cell polarization and directed cell migration is complex, given that a cell needs to integrate numerous signals arising from different spatial orientations to decide in which direction to move. In lymphocytes, the signals that guide the cell in making these decisions arise from chemokine activation of heptahelical G protein-coupled receptors (GPCR) linked to G␣i proteins. Directional migration, however, requires the activity of G␤␥, but not G␣, proteins (1, 2). In addition, Rho family guanosine triphosphatases (GTPases), the phosphoinositide 3-kinases, and possibly extracellular receptor kinases each play important roles in generating the cell polarity and cytoskeletal reorganization required for directional migration (3-7).Migration to chemokine gradients is dose dependent. Chemotaxis occurs at relatively low concentrations of chemokines, but at higher chemokine concentrations, cells become paralyzed and no longer migrate toward the chemoattractant source. The mechanisms by which cells are paralyzed at high chemokine concentrations are not clear but may involve agonist-dependent desensitization and receptor endocytosis mediated by G proteincoupled receptor kinases (GRKs) and arrestins. GRKs phosphorylate serine and threonine residues in the C terminus and intracellular loops of GPCRs, allowing for the association of arrestins that act to prevent heterotrimeric G␣␤␥ protein association with a...

Non-visual GRKs: are we seeing the whole picture?

“…Comparison of the chemotactic responses of T and B cells isolated from GRK5 2/2 or GRK6 2/2 mice indicated that GRK6-deficient cells were impaired in their ability to respond to the chemokine CXCL12 (a selective ligand for the chemokine receptor CXCR4), whereas GRK5-deficient cells were unaffected [43]. The potential role of GRK6 in dopamine receptor regulation has also been highlighted recently in GRK6 2/2 mice, which showed supersensitivity to psychostimulant drugs [44].…”

Emerging Roles of β-Arrestins

“…37 Also, splenocytes derived from β-arrestin 2 knockout mice showed decreased CXCL12-induced migration. 38 Our group recently published a novel role for β-arrestin 1 in cellular migration and metastasis. 39 In colorectal carcinoma cells, prostaglandin E 2 stimulation of the EP4 receptor, a GPCR, leads to increased cellular migration in vitro.…”

Fenoterol, a β2-adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through β-arrestin-2 in THP-1 cell line

“…For example, β-arrestin-2-dependent stabilization of cytosolic IκBα and inhibition of NF-κB activation following LPS stimulation are essential for rapid and sufficient production of NO in response to microbial attack [14] . Moreover, there is accumulating evidence that β-arrestin-2, which is expressed abundantly in the spleen, is functionally involved in some important immune responses, such as regulation of lymphocyte chemotaxis and homing [24,25] . In the present study we used RNA interference against β-arrestin-2 to test its role in anti-inflammatory effects stimulated by β 2 -AR.…”