Control Of G Protein Signaling: Role Of The RGSs

National Institute Of Allergy And Infectious Diseases

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Abstract

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gi signaling, whose magnitude and kinetics are governed by RGS protein/Gi interactions. RGS proteins typically limit Gi signaling by reducing the duration that Gi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. We have shown that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gi2/RGS protein interactions both limit and facilitate Gi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance. Transendothelial and interstitial lymphocyte migration revealed by intravital microscopy. Actin is essential for many cellular processes including cell motility. Yet F-actin dynamics during lymphocyte transendothelial migration (TEM) and interstitial migration have not been visualized. We used high-resolution confocal intravital imaging with LifeAct-GFP bone-marrow reconstituted mice, which allowed visualization of lymphocyte F-actin dynamics in vivo. We found that nave lymphocytes preferentially cross high endothelial venules (HEVs) using the paracellular rather than the transcellular route. During both modes of transmigration F-actin levels rise at the lymphocyte leading edge as the cell engages the TEM site. Once the lymphocytes breach the endothelium, they briefly reside in HEV pockets prior to crossing into the parenchyma. During interstitial migration dynamic actin-based protrusions rapidly form and collapse to help drive motility. Using a panel of inhibitors, we established roles for actin regulators and myosin II in lymphocyte TEM. This study provided unprecedented views of lymphocyte TEM and interstitial migration in vivo. We established methodology to assess F-actin and myosin IIA dynamics during B cell and neutrophil migration. 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. Activated Gi recruited Elmo1 to the plasma membrane. These studies support a prominent role for a Galpha i/Elmo1/Dock2/Rac/Wave regulatory complex signaling pathway in the initial F-actin response of B cells to CXCL13. Using ICAM-1 and chemoattractant coated imaging chambers along with an agarose overlay, we imaged the motility of LifeAct GFP and Myosin IIA-GFP expressing B-lymphocytes and neutrophils. In the under agarose assays the neutrophils migrated much more vigorously than did the B cells, however, a 6 h exposure to LPS significantly improved B cell migration. In both B cells and neutrophils F-actin accumulated at the leading cell edge and in the pseudopods of the migrating cells. Inactivating Gi nucleotide exchange markedly impaired the motility of both the nave and 6 h LPS activated B cells. 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 gene encoding regulator of G-protein signaling1 (Rgs1) represents one of the most up-regulated gene in tissue resident memory (TRM) cells. A major function of Rgs1 in lymphocytes is to regulate chemoattractant receptor signaling by limiting the duration that Gi subunits remain GTP bound and capable of activating downstream effectors. We hypothesized that Rgs1 affects the formation and retention of TRM CD8+ T cells. We found that following oral Listeria monocytogenes-OVA infection, antigen-specific Rgs1-/- CD8+ OT-I T cells show an elevated T-bet/Eomes mRNA expression ratio and an impaired differentiation into memory precursor effector cells (MPEC) in the intestinal mucosa. Consequently, Rgs1-/- OT-I CD8+ TRM cells become underrepresented in the intestinal mucosa at day 30 post-infection. While Rgs1+/+ CD8+ T cells clear the pathogen following re-infection, antigen-specific Rgs1-/- CD8+ TRM cells do not efficiently clear the pathogen following reinfection. These date indicate that Rgs1 expression in OT-I CD8+ T cells promotes local accumulation of MPEC and their functional differentiation into functional TRM cells (this was a collaboration with Christoph Mullers laboratory). Lymphocyte Function-Associated Antigen 1 (LFA-1) binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells, and helps coordinate T cell migration, adhesion, and activation. LFA-1 is activated during chemokine and TCR receptor engagement through inside-out signaling via T cell and chemoattractant receptor mediated phosphoinositide 3-kinase (PI3K) activation, and its product PI(3,4,5)P3 (PIP3). To evaluate roles of PIP3-binding proteins in LFA-1 activation, we designed a retroviral library encoding CRISPR/Guide RNAs targeting known and potential PIP3-binding proteins. Using this library to screen for effects on ICAM-1-binding of Cas9-expressing primary mouse T cells, we identified multiple proteins regulating LFA-1-mediated adhesion, including the RAP1/RAS GTPase-activating protein, RASA3. We found RASA3 to be a critical negative regulator of LFA-1 activation that is inhibited by PI3K activity. Imaging experiments identified a strong co-localization between RASA3 and Gi, the major transducer of chemoattractant receptor signaling and RASA3. RASA3 overexpression in HeLa cells caused a marked increase in cytoneme formation. T cells lacking RASA3 exhibited elevated ICAM-1-binding associated with increased RAP1 activation, defective lymph node entry and egress, and impaired help for T-dependent immunization. Our results have uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function (this study is an ongoing collaboration with Pam Schwartzberg's laboratory).