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.
Highlights
G protein– coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients
The G␣i/o family has been strongly implicated in chemotactic signaling because pertussis toxin (PTX; previously known as lymphocytosis-promoting factor) from Bordetella pertussis, which blocks the activation of G␣i/o subunits by G protein– coupled receptors (GPCRs), inhibits the chemotaxis of macrophages and other phagocytes (9 –11)
We used knockout mouse models to explore the roles of the various G␣ and G subunits shown in Fig. 1B, except Gnas (G␣s), in transducing gradients of the chemoattractant complement C5a into stimulated motility and directed cell migration using the -Slide Chemotaxis chamber [16, 18] and time-lapse, phase-contrast microscopy
Summary
RNA sequence analysis of purified resident peritoneal F4/80ϩ cells (macrophages) revealed that Gnai (G␣i2) and Gnai (G␣i3) of the G␣i/o family of G␣-subunits, as well as Gnas (G␣s) and members of the G␣q/G␣11 (Gnaq, Gna, and Gna15) and G␣12/G␣13 (Gna and Gna13) families, are expressed, as well as complement C5a receptor 1 (as known as CD88), encoded by C5ar (Fig. 1, A and B). Davignon et al [14] showed that Gna is not required for normal hematopoiesis but found that complement C5a-induced Ca2ϩ transients, averaged from at least five cells, were markedly decreased in thioglycolate-elicited peritoneal macrophages from Gna15Ϫ/Ϫ mice, whereas responses to ATP or UTP were similar to WT macrophages. Application of complement C5a induced rapid and robust lamellipodial membrane protrusions in WT (Fig. 9A and Video macrophages (Video S15), whereas no increase in intracellular [Ca2ϩ] is seen in Gna15Ϫ/Ϫ macrophages (see the Cal-520 fluorescence intensity trace in Fig. 9A and Video S16). Both cell velocity and chemotactic efficiency were significantly decreased in Gnb2Ϫ/Ϫ macrophages compared with WT controls
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