Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

Abstract

G protein–coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gβ, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gβ subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2−/− mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2−/− macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gβ1) deletion was lethal, but mice lacking Gnb2 (Gβ2) were viable. Gnb2−/− macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gβ2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms

“…Methods for the isolation and handling of mouse resident peritoneal macrophages have been previously described ( 31 , 54 ). In brief, mice were sacrificed by an overdose of isoflurane followed by cervical dislocation, and the peritoneal cavity was lavaged (2 × 4.5 ml) via a 24-G plastic catheter (B. Braun) using ice-cold Hank’s balanced salt solution without Ca 2+ or Mg 2+ (14175-046; Gibco, Life Technologies) ( 55 ).…”

Gα12 and Gα13: Versatility in Physiology and Pathology

“…In macrophages, complement C5a couples to Gα 12/13 to activate Rho GTPases and tail retraction in migrating cells. Macrophages without Gα 12/13 show complete chemotaxis but increased migration speed and a moderately impaired tail contraction ( van den Bos et al, 2020 ). Thrombin, a major platelet activator, selectively induces the expression of CD36, a plasma membrane fatty acid transporter, and foam cell formation of RAW264.7 cells through PAR1-Gα 12 signaling and facilitates atherosclerosis ( Raghavan et al, 2018 ).…”

Alphataxin, an Orally Available Small Molecule, Decreases LDL Levels in Mice as a Surrogate for the LDL-Lowering Activity of Alpha-1 Antitrypsin in Humans

“…Endocytosis at the trailing edge of the migrating cell stimulates NF-κB phosphorylation, and release of the trailing edge of the cell from the tissue matrix, a situation that propels the cell forward ( Bristow et al, 2008 ; Bristow and Winston, 2021 ). This process, in common with many other cell surface proteinase/proteinase inhibitor pairs, is constitutive and provides a physical mechanism for CD4 + T lymphocytes to sample the environment for nutrients and toxins, induce nuclear signaling, and promote cellular locomotion ( Chu et al, 1994 ; Elia et al, 2007 ; van den Bos et al, 2020 ).…”

“…Antibiotic activity. The Clinical and Laboratory Standards Institute (CLSI) approved protocol for the Kirby-Bauer Disk Test was performed to compare the antibiotic activities of D-ampicillin with Alphataxin(Turnidge and Paterson, 2007; Performance Standards for Antimicrobial Susceptibility…”