We have shown that RGS proteins modulate signaling through a variety of G-protein coupled receptors including chemokine receptors. Chemokine receptors signal predominantly by triggering G alpha i nucleotide exchange. Humans and mice have three Gi isoforms although Gi alpha2 (encoded by Gnai2) and Gi alpha3 (encoded by Gnai3) predominate in lymphoid cells. We have found that Gnai2 -/- T and B cells have severe defects in chemokine-receptor signaling while Gnai2 +/- T and B cells exhibit modest defects. In vivo, the Gnai2-/- B cells fail to properly access lymph node follicles and the Gnai2-/- T cells failing to properly enter the T cell zone. We have studies the immune system with a focus on immune cell trafficking in various RGS deficient mice. We have supplemented these studies with in vitro studies using mRNA knock-downs as well as over expression studies using RGS proteins fused to a fluorescent marker. The recognition of the importance of both G-protein signaling and RGS proteins in the regulation of lymphocyte responses to chemokines has led us some of our studies to hematopoietic cell types. RGS13 is strongly expressed in both human and mouse germinal center B cells and is often found highly expressed in human B cell lymphomas of germinal center origin. Rgs13 is also well expressed in mast cells and in adult T cell leukemia cells. To facilitate our studies of RGS13 and to provide a potential resource for in vivo imaging endogenous germinal center B cells we have generated mice where GFP (green fluorescent protein) has been knocked-in (KI) into the Rgs13 locus replacing the RGS13 coding sequence. Flow cytometry revealed high levels of GFP expression in germinal center centroblasts and centrocytes. Intravital microscopy documented the presence of brightly GFP positive cells in the interfollicular region and at the T-B border within a day of immunization. Later strong expression is observed in B cells located in germinal centers. Immune phenotyping of these mice revealed a minor defect in early B cell development with a normal peripheral lymphoid compartment with the exception of a 25% expansion in the number of Peyers patches. Upon immunization the Rgs13-/- mice exhibited an enhanced extra-follicular antibody response, an exaggerated germinal center response, and a disturbance of germinal center architecture. Analysis of mRNA expression in Rgs13GFP positive B cells revealed a marked enrichment in germinal center specific genes as well as genes involved in cell proliferation. Analysis of irradiated mice reconstituted with 1:1 mix of wild type and Rgs13-/- bone marrow revealed an expansion of Rgs13-/- GC B cells at the expense of wild type cells. These studies are aimed at understanding the role RGS13 plays in germinal center B cell function. Rgs14 is well expressed in lymphocytes and initially targeting in mice indicated that its absence resulted in embryonic lethality at the two cell stage. To analyze the role of Rgs14 in immune cells we created Rgs14LoxP mice. However, deletion of Rgs14 was not deleterious so we have analyzed mice globally lacking Rgs14 expression. Initial analysis of the immune system of these mice was unrevealing. Rgs19 is well expressed in B lymphocytes and less so in T cells. It is also well expressed in stem cells and hematopoietic progenitors. GFP has been inserted into the RGS19 locus and appropriated targeted mice identified. Flow cytometry revealed that GFP is well expressed in mature B and T cells and in progenitors in the bone marrow. Consistently the Rgs19 GFP KI mice had an enlarged spleen and thymus compared to wild type mice. Chemotaxis experiments comparing wild type and RGS19-/- lymphocytes indicate significant increases to a panel of chemokines with the KO B cells. Serum levels of IgM, IgG2a, and IgG2b are elevated in these mice. Current studies are aimed understanding the pathogenesis of the splenomegaly and enhanced B cell responsiveness in the Rgs19-/- mice. In bone marrow mixed chimeras the Rgs19-/- deficient B cells and neutrophils expand preferentially. Intriguing the wild type cells also expand in the mixed chimera mice suggesting that some bone marrow derived Rgs19-/- cell is responsible for the expansion. As another approach to assessing the role of RGS proteins we have begun the analysis of mice with a Gnai2 G184S knock-in obtained from Richard Neubig (University of Michigan). This mutation renders Gi alpha2 resistant to the effect of RGS proteins. The Gnai2 G184S KI mice possess a striking phenotype verifying the overall important of RGS proteins in G-protein regulation. They have a marked reduction in peripheral T cells;an increase in mature T cells in the thymus;a thymocyte egress defect;an absence of many peripheral lymph nodes;a disorganized spleen architecture;splenomegaly;abnormal lymphocyte responses to chemokines;and reduced serum levels of IgG3, but increased levels of Ig2b. We have shown that neutrophils with a genomic knock-in of a mutation that disables RGS protein-Galphai2 interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a non-lethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gi alpha activation helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent. It has been increasing evident that G-proteins and G-protein signaling have role in cells beyond their known roles as receptor transducers. We have begun a study of the role of G-proteins in macrophage phagocytosis. Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors whether such signaling pathways affect phagocytosis is contentious. We have shown that Galpha i nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin and Galphai proteins to cell protrusions, phagocytic cups, and early phagosomes. Furthermore, macrophages recovering from F-actin depolymerization exhibit a synchronized re-appearance of F-actin and Gαi at cell protrusions. Inhibiting GDP-Galpha i nucleotide exchange or pharmacologically interrupting Gβγsignaling impairs phagocytosis while favoring the duration that Galpha i remains GTP bound promotes it. Macrophages lacking Galpha i2 or Galpha i3, or with lowered expression of the Galpha i nucleotide exchange factor Ric-8A have reduced phagocytic capacities. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets.

Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2013
Total Cost
$514,781
Indirect Cost
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Branham-O'Connor, Melissa; Robichaux 3rd, William G; Zhang, Xian-Kui et al. (2014) Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3). J Biol Chem 289:10738-47
Hwang, Il-Young; Hwang, Kyung-Sun; Park, Chung et al. (2013) Rgs13 constrains early B cell responses and limits germinal center sizes. PLoS One 8:e60139
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Cho, Hyeseon; Park, Chung; Hwang, Il-Young et al. (2008) Rgs5 targeting leads to chronic low blood pressure and a lean body habitus. Mol Cell Biol 28:2590-7