Mast cells (MCs), granulocytes, and lymphocytes are integral to the development of an allergic response. Allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of G protein-mediated signal transduction in immune cells, with a focus on GPCR-mediated trafficking of leukocytes to sites of allergic inflammation. GPCRs activate a core pathway of heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GPCR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in allergic inflammation. A major area of investigation is the recruitment of inflammatory leukocytes to sites of inflammation. Chemokines are a major class of compounds acting on leukocyte GPCRs, which orchestrate immune cell trafficking, and RGS proteins including RGS5, RGS13, and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. In FY18, we continued our phenotypic characterization of patients with undefined immunodeficiencies and novel mutations in G proteins and/or RGS proteins are being characterized in collaborative studies with Drs. Orange and Su. 3 brothers in a single family with combined immunodeficiency were found to carry biallelic mutations in RGS10. We discovered that RGS10 is expressed in both human B and T cells. Patient-derived B and T cells from these patients exhibit profoundly impaired chemotaxis in response to chemokines despite having comparable RGS10 expression. Molecular studies, which are ongoing, have determined that the mutations may affect RGS10 phosphorylation and induce protein mislocalization, which could result in a gain of function. IN FY18, we defined a role for RGS5 in neutrophil trafficking. RGS5 was expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than wild-type counterparts following acute respiratory virus infection and also in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12, but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP.
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