G Protein-coupled receptor kinase-6 interacts with activator of G protein signaling-3 to regulate CXCR2-mediated cellular functions.

The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.

G-protein signaling modulator-3, a gene linked to autoimmune diseases, regulates monocyte function and its deficiency protects from inflammatory arthritis.

Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, collagen antibody-induced arthritis (CAIA) was induced in Gpsm3-/- and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3-/- mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis.

Proteomic surveillance of autoantigens in patients with Behcet's disease by a proteomic approach.

To promote an understanding of autoimmunity in BD, we surveyed autoAgs in patients with BD and investigated the prevalence and clinical significance of the identified autoAbs. Specifically, proteins, extracted from peripheral blood mononuclear cells and separated by 2DE, were subjected to WB, using five serum samples from patients with BD. The detected candidate autoAgs were identified by mass spectrometry. As a result, 17 autoantigenic spots were detected by the 2DE-WB, out of which eight spots were identified. They are enolase-1, cofilin-1, vimentin, Rho-GDI beta protein, tubulin-like protein, and actin-like proteins. The autoAbs to one of the identified proteins, cofilin-1, were investigated by WB using a recombinant protein in 30 patients with BD, 35 patients with RA, 32 patients with SLE, and 16 patients with PM/DM. The autoAbs to cofilin-1 were detected by WB in four (13.3%) of the 30 patients with BD, five (14.3%) of the 35 patients with RA, two (6.3%) of the 32 patients with SLE, and eight (24.2%) of the 33 patients with PM/DM. Our data indicate that the generation of autoAbs to cofilin-1 may reflect common immunological disorders in BD, RA, and PM/DM. Our data would help understanding of the immunopathology of BD. In addition, the proteomic approach would be a useful way to investigate autoAgs.

Antibodies in patients with neuropsychiatric systemic lupus erythematosus.

OBJECTIVE: To investigate a target for antibodies in patients with neuropsychiatric systemic lupus erythematosus (NPSLE). BACKGROUND: Pathogenesis of NPSLE may be related to autoantibody-mediated neural dysfunction, vasculopathy, and coagulopathy. However, very few autoantibodies are sensitive and specific to NPSLE because the neuropsychiatric syndromes associated with SLE are diverse in cause and presentation. METHODS: We identified antibodies against brain antigens in the sera of 7 patients with NPSLE and 12 healthy controls by 2-dimensional electrophoresis, followed by Western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS), using rat brain proteins as the antigen source. RESULTS: Six antibodies were detected in patients with NPSLE. One of these 6 antibodies was found in antibodies against Rab guanosine diphosphate dissociation inhibitor alpha (alphaGDI) (which is specifically abundant in neurons and regulates synaptic vesicle exocytosis) in patients with NPSLE with psychosis. We tested more samples by 1-dimensional immunoblotting of human recombinant alphaGDI. Positivity of the anti-alphaGDI antibody was significantly higher in patients with NPSLE with psychosis (80%, 4 of 5) than in patients with NPSLE without psychosis (0%, 0 of 13), patients with systemic lupus erythematosus without neuropsychiatric symptoms (5.3%, 1 of 19), patients with multiple sclerosis (0%, 0 of 12), patients with infectious meningoencephalitis (0%, 0 of 13), patients with polyneuropathy (0%, 0 of 10), patients with psychotic syndromes (0%, 0 of 10), and healthy controls (0%, 0 of 12). CONCLUSIONS: We propose that the anti-Rab guanosine diphosphate dissociation inhibitor alpha antibody is a candidate for further exploration as diagnostic marker of psychosis associated with neuropsychiatric systemic lupus erythematosus.

CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex.

In response to invading microorganisms, macrophages engage in phagocytosis and rapidly release reactive oxygen species (ROS), which serve an important microbicidal function. However, how phagocytosis induces ROS production remains largely unknown. CARD9, a caspase-recruitment domain (CARD)-containing protein, is important for resistance to fungal and bacterial infection. The mechanism of CARD9-mediated bacterial clearance is still mostly unknown. Here we show that CARD9 is required for killing intracellular bacteria in macrophages. CARD9 associated with the GDP-dissociation inhibitor LyGDI in phagosomes after bacterial and fungal infection and binding of CARD9 suppressed LyGDI-mediated inhibition of the GTPase Rac1, thereby leading to ROS production and bacterial killing in macrophages. Thus, our studies identify a key pathway that leads to microbe-elicited ROS production.

Rho GTPase-mediated pathways in mature CD4+ T cells.

Effective immune responses require the appropriate activation and differentiation of peripheral CD4(+) T cells. These processes need to be followed by the timely elimination of the responding T cells in order to restore T cell homeostasis. Defects in the appropriate regulation of T cell activation, expansion, and survival underlie the pathogenesis of many autoimmune disorders including SLE. The molecular machinery employed by T cells to properly control these processes and prevent the onset of autoimmunity has not been fully elucidated. Rho GTPases (which include the Rac, Cdc42, and Rho subfamilies) are molecular switches that control a wide range of cellular processes. Their fundamental role in biology is due to their ability to regulate both cytoskeletal dynamics and a large number of signal transduction pathways. Activation of Rho GTPases is now recognized as a key event in the coordination of immune responses and, particularly, in the activation of T cells. In this review, we will first provide an overview of the role of Rho GTPase-mediated pathways in mature CD4(+) T cells and then we will discuss recent studies, which suggest that deregulation of these pathways may play a role in the pathogenesis of SLE.

Altered cellular immunity in transgenic mice with T cell-specific expression of human D4-guanine diphosphate-dissociation inhibitor (D4-GDI).

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.

High-efficiency solid-phase capture using glass beads bonded to microcentrifuge tubes: immunoprecipitation of proteins from cell extracts and assessment of ras activation.

We have bonded glass microbeads (425-600 microm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A- or G-agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G-agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20-30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A- or G-agarose beads.

Production and characterization of isotype-specific monoclonal antibodies to bovine brain Rab GDI.

Small GTPases of the Rab family play a key role in controlling vesicular transport, and the Rab GDP-dissociation inhibitor (GDI) is a regulatory protein for the Rab proteins. Here we report the production and characterization of isotype-specific monoclonal antibodies (MAbs) to Rab GDI. Rab GDI was purified from bovine brain in several steps of column chromatography and was injected into BALB/c mice intraperitoneally. The resulting MAbs specifically recognized a single protein band of 55 kDa, which comigrates with purified bovine Rab GDI. To localize Rab GDI, we processed cells from different sources for indirect immunofluorescence microscopy. Interestingly, the MAb stained cytosol and vesicular structures in brain cells, whereas it predominantly stained cytosol in nonbrain cells. Next, we investigated the cross-reactivities of brain Rab GDI from some mammals. The immunoreactive bands on Western blots appeared to be the same in molecular mass, 55 kDa, in all mammalian species tested including human. In summary, we produced a panel of MAbs that are GDI-alpha/1 form-specific and we believe that the MAbs will be valuable tools in elucidating the function of Rab GDI isoforms.