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. G protein alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. 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 intrinsic GTPase activity, a property that promotes deactivation of the signaling pathway. 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 (FY) 23, we characterized the phenotype of patients with undefined immunodeficiencies and novel mutations in G proteins in collaborative studies with Dr. Su. During G-protein coupled receptor (GPCR) signaling, heterotrimeric G-proteins undergo cycles of GDP/GTP exchange and hydrolysis with activation or deactivation of downstream effector signals, coordinated with dissociation and reassembly of their subunits with GPCR. We described 17 patients from 16 kindreds with rare heterozygous mutations in GNAI2 that impeded GTP hydrolysis by Galphai2, thereby augmenting Glaphai2s inhibitory signaling. The patients had congenital malformations in diverse tissues and infection susceptibility with immunodyregulation caused by defective cell migration. More potent activating mutations also impeded G-protein reassembly to block subsequent GPCR responses to chemokines. Our findings explain the paradoxical biological effects that result from inhibiting the cycling of Gi between its active and inactive forms for Gi association with GPCR in humans. In a second area of investigation, we further elucidated the functions of RGS4 in an experimental model of asthma. RGS4 has documented interactions with multiple intracellular proteins other than G-alpha subunits, including STAT1, PI3 kinase, and b'-COP. Knockin mice expressing a GAP inactive mutant RGS4 (N128A) had preserved RGS4 expression and exhibited increased airway hyper-responsiveness in a model of allergic airway inflammation compared to either WT or RGS4 KO mice. These findings reveal that RGS4 has GAP-independent functions in vivo.