Heterotrimeric G Protein Signaling In Allergic Inflammation

National Institute Of Allergy And Infectious Diseases

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Abstract

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 RGS10, RGS13, and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. In fiscal year 19, we continued to characterize the phenotype of patients with undefined immunodeficiencies and novel mutations in G proteins and/or RGS proteins in collaborative studies with Drs. Chinn, Orange and Su. We characterized the clinical and molecular phenotype of three siblings from one family, who presented with short stature and recurrent gastrointestinal and sinopulmonary infections. Exome sequencing (ES) revealed that each affected sibling carried novel, uncharacterized variants of RGS10 (delE163 and A171S). We also detected and characterized variants of PIK3CD in this cohort; however, none of these siblings manifested the autoimmunity or malignancy typical of the clinical disease trait associated with this locus; instead the affected subjects displayed growth hormone deficiency, hypergammaglobulinemia, and normal naive T cell numbers and proliferation, none of which are associated with isolated PIK3CD defects. Lymphocytes from all three siblings had strongly impaired migration to chemoattractants and abnormal localization within lymph nodes. Although the GAP activity of each RGS10 variant was intact, each protein exhibited aberrant phosphorylation by protein kinase A (PKA); this was associated with increased cytosolic and cell membrane localization and activity compared to the WT protein. We propose that the RGS10 delE163 and A171S mutations lead to mis-localization of the RGS10 protein, resulting in a unique syndrome of short stature and immunodeficiency due to irregularities in metabolism and lymphocyte trafficking. This is the first report of a human genetic disorder linked to rare variants in an RGS family member. IN FY19, we continued our longstanding collaboration with Dr. Kasanwneh to characterize RGS protein expression and function in platelets. There are 3 different RGS isoforms that are expressed in platelets including RGS16. We used a genetic knockout mouse model approach to examine the role(s) of RGS16 in platelet activation. We observed that agonist-induced platelet aggregation, secretion, and integrin activation were much more pronounced in platelets from the Rgs16-/- mice relative to their wild-type littermates. Bleeding times were shortened in Rgs16-/- mice and they were more susceptible to vascular injury-associated thrombus formation than the controls.