In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein ␣ (G␣) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G␣ i function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G␣ i , thus preventing its GEF activity for G␣ i . Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G␣ i expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.

RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5-E17.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E14.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Gi(alpha) and Gq(alpha) and it attenuated angiotensin II-, endothelin-1-, sphingosine-1-phosphate-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation.

Signaling by G protein-coupled receptors coupled to Galpha(i) assists in triggering lymphocyte movement into and out of lymph nodes. Here, we show that modulating the signaling output from these receptors dramatically alters B cell trafficking. Intravital microscopy of adoptively transferred B cells from wild-type and Rgs1-/- mice revealed that Rgs1-/- B cells stick better to lymph node high endothelial venules, home better to lymph nodes, and move more rapidly within lymph node follicles than do wild-type B cells. In contrast, B cells from Gnai2-/- mice enter lymph nodes poorly and move more slowly than do wild-type B cells. The Gnai2-/- mice often lack multiple peripheral lymph nodes, and their B cells respond poorly to chemokines, indicating that Galpha(i1) and Galpha(i3) poorly compensate for the loss of Galpha(i2). These results demonstrate opposing roles for Rgs1 and Gnai2 in B cell trafficking into and within lymph nodes.