Control of G-protein Signaling: Role of RGSs

National Institute Of Allergy And Infectious Diseases

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Abstract

We established methodology to assess F-actin and myosin IIA dynamics in B cells. Using a flow-based assay, we examined the chemoattractant mediated signaling pathway that induced F-actin formation in resting B cells. F-actin levels increased within 5 seconds and peaked at 20 seconds after CXCL13 exposure. F-actin formation depended upon increases in both branched and linear F-actin, Galpha i protein nucleotide exchange, Dock2, and Rac activation. Inhibiting Erk or Src activation, or Myosin IIA reduced, but did not eliminate CXCL13 induced F-actin. Inhibiting Gbeta/gamma signaling slightly reduced the induction of F-actin, while inhibition of BTK or RhoA had no effect. Using ICAM-1 and chemokine coated imaging chambers along with an agarose overlay, we imaged the motility of LifeAct GFP and Myosin IIA-GFP expressing B-lymphocytes. F-actin accumulation at the leading cell edge and in the pseudopods of the migrating cells. Inactivating Gi nucleotide exchange markedly impaired B cell motility. Myosin IIA weakly accumulated at the leading edge and strongly in the pseudopods. Myosin IIA also accumulated on the lateral edge of turning cell on the side opposite the direction of the turn. Intravital imaging of myosin IIA-GFP B cells confirmed the dynamic behavior of Myosin-IIA during transendothelial and interstitial migration of B cells in vivo. These studies are extending of the signaling pathways and molecules that control B cell movement in vivo. The responsible signaling events driving the transient F-actin increase depended upon Gi2/3-signaling, the PI-3 kinase/AKT pathway, ERK activation, phospholipase C activity, and Dock2 mediated Rac1/2 activation. AKT substrate and pT58 WNK1 immunoblotting identified WNK1 (with no lysine kinase 1) as a potential early AKT effector. Verifying its importance, treating B cells with specific WNK inhibitors reduced pAKT and pERK activation, disrupted F-actin dynamics, and impaired B cell polarity, motility, and chemotaxis. CRISPR/Cas9 gene editing of WNK1 in a murine B cell line confirmed the inhibitor data and suggested that WNK1 contributes to B cell proliferation. A one-time administration of a WNK inhibitor to mice transiently reduced lymph node B cell motility and polarity in vivo. These results indicate that WNK1 signaling maintains B cell responsiveness to chemokines and suggests that pharmacological inhibition of WNK1 may have untoward effects on humoral immunity. The small GTPase Rap1 regulates lymphocyte integrin affinity, a critical step in the arrest of lymphocytes on high endothelial cells (HEVs) and in transendothelial migration. Rap1 guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) regulate Rap1 by controlling its GTP/GDP binding status. Loss of the Rap1 GAP Rasa3 in platelets caused thrombocytopenia while loss in developing thymocytes caused T cell lymphopenia. We investigated how Rasa3 deficiency impacted B cell trafficking by conditionally deleting Rasa3 in mouse B cells. A severe maldistribution of B cells resulted. The Rasa3 deficient B cells (Rasa3 KO) overpopulated the spleen, thymus, and peritoneal cavity, but underpopulated the blood, lymph nodes (LNs), and Peyer’s Patches (PPs). The Rasa3KO B cells rapidly adhered to LN HEVs, yet poorly transmigrated. Few Rasa3KO B cells bound to or crossed PP HEVs, and those that entered rapidly egressed. PPs contained less than 10% of their usual number of B cells. The analysis of mixed chimeras supported these results. Basally the Rasa3KO B cells avidly bound soluble MAd-CAM-1 and ICAM-1, while CXCL13 stimulation minimally upregulated the binding. The Rasa3 deficient B cells had basally elevated Rap1-GTP and Ras-GTP levels and CXCL13 stimulation elicited heightened levels compared to controls. Surprisingly B cell proliferation in response to a panel of stimuli was reduced yet cell viability was enhanced. The Ras3 B cell KO mice had low serum immunoglobulins levels of IgG1, IgG3, and IgA. Rasa3 limits basal GTP bound Rap1 allowing B cells to properly traffic into and through lymphoid organs and tissues. In addition, Rasa3 limits basal GTP-bound Ras augmenting B cell survival, partially explaining the splenomegaly observed in these mice. Bulk RNA sequencing data was analyzed using B cells prepared from the spleens of 12 Rasa3 B cell KO mice and 12 control mice. Six of each genotype were cultured for 4 hours in media alone and the remainder cultured with CXCL13. Ingenuity pathway analysis revealed an increase in Ras target genes in the Rasa3 KO B cells both in the non-stimulated and in the CXCL13 stimulated B cells. We also noted a strong reduction in IgA transcripts in the Rasa3 KO B cells consistent with the loss of mucosal B cells in these mice. Imaging of a Rasa3-GFP in HeLa cells revealed Rasa3 localization at the plasma membrane. Plasma membrane localization was reduced by treating the cells with the PI3K inhibitor wortmannin or pertussis toxin. Raising intracellular calcium levels also reduced the membrane localization. Deletion of the N-terminal C2 domains did not affect the plasma membrane localization, however a C-terminal deletion beginning prior to the PH domain markedly reduced it. A K599Q/K600Q/R601Q Rasa3 protein, which no longer binds phosphoinositols in membranes, lost plasma membrane targeting and localized in the cytosol and nucleus. Confocal, TIRF, and FILM microscopy revealed Rasa3 co-localization with GTP bound Gαi2. Co-immunoprecipitation experiments documented that GTP-bound Gαi2 interacted with the full length Rasa3, 1-280 N-terminal deleted Rasa3, but poorly with either 1-550 N-terminal or a 550-834 C-terminal deletion. Alpha-fold and molecular dynamics mapped the interaction between GTP bound Gαi2 and Rasa3 to the C-terminal portion of Rasa3 and identified 4 amino acids likely to mediate the interaction. These 4 amino acids were evolutionarily conserved, 3 of the 4 were conserved between Rasa2 and Ras3, but they were not present in Rasa1. Mutating them in Rasa3 slightly reduced the plasma membrane localization and led to an inability of the mutated protein to interact with GTP bound Gαi2. A molecular dynamics simulation using the 1-550 predicted Rasa3 protein explained its poor interaction with GTP bound Gαi2 as the deletion disrupted the conformation of the C-terminal portions of Rasa3. Expression of GTP bound Gαi2 recruited K599Q/K600Q/R601Q Rasa3 protein from the cytosol to the plasma membrane. Interestingly most Epstein Barr transformed human B cell lines lack Rasa3 expression suggesting that the transformation process may directly target Rasa3 for downregulation. Permanent expression of Rasa3-mScarlet in an EBV transformed human B cell line revealed that like HeLa cells Rasa3 resided predominately at the plasma membrane. Western blotting showed good expression of the Rasa3 protein in the transfected cell lines. Functional studies are in progress to assess the impact of expressing Rasa3 and to assess how EBV transformation downregulates Rasa3 mRNA and protein expression. B cells express multiple RGS proteins including Rgs1, Rgs2, Rgs3, Rgs9, Rgs10, Rgs11, Rgs12, Rgs13, Rgs14, Rgs18, and Rgs19. A cluster of Rgs genes, Rgs1, Rgs2, Rgs13, and Rgs18 is located on chromosome 1 in both mice and humans. Because of functional redundancy the loss of an individual Rgs protein often results in a minimal phenotype. To better understand the role of the Rgs genes clustered on chromosome 1 we have used CRISPR/Cas9 gene editing of mouse embryos to simultaneously target Rgs1, Rgs2, Rgs13, Rgs18. 3 injections using 8 sgRNA for deletion, generated 55 pups, and 3-4 promising candidate mice with multiple knock-outs in RGS. One injection using 4 sgRNA for insertion-deletion mutations (indels), generating 9 pups 2 promising candidate mice with multiple indels. The candidate mice have been back crossed to wild type mice to assess success of the targeting. Mice with a single Rgs18 knockout, double knockout of Rgs1 and Rgs13, double knockout of Rgs2 and Rgs13, and triple knockout of Rgs1, Rgs2, and Rgs13 have been found and are being crossed to generate mice homozygotic mice for these gene knockouts and used for phenotyping. ES cells from the triple knock-out strain will be used to generate a mouse lacking all four targeted genes.