Human cells contain four homologous Ras proteins, but it is unknown whether each of these Ras proteins participates in distinct signal transduction cascades or has different biological functions. To directly address these issues, we assessed the relative ability of constitutively active (G12V) versions of each of the four Ras homologs to activate the effector protein Raf-1 in vivo. In addition, we compared their relative abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell migration. We found a distinct hierarchy between the four Ras homologs in each of the parameters studied. The hierarchies were as follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>> N-Ras > Ha-Ras; for focus formation, Ha-Ras >/= Ki-Ras 4A >>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras 4A >/= N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras 4A = no migration. Our results indicate that the four Ras homologs significantly differ in their abilities to activate Raf-1 and induce distinctly different biological responses. These studies, in conjunction with our previous report that demonstrated that the Ras homologs can be differentially activated by upstream guanine nucleotide exchange factors (Jones, M. K., and Jackson, J. H. (1998) J. Biol. Chem. 273, 1782-1787), indicate that each of the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also suggest for the first time that the distinct and likely cooperative biological functions of the Ki-ras-encoded Ki-Ras 4A and Ki-Ras 4B proteins may help explain why constitutively activating mutations of Ki-ras, but not N-ras or Ha-ras, are frequently detected in human carcinomas.

Sphingosine 1-phosphate (S1P) in blood, lymph, and immune tissues stimulates and regulates T cell migration through their S1P1 (endothelial differentiation gene encoded receptor-1) G protein-coupled receptors. We show now that S1P1Rs also mediate suppression of T cell proliferation and cytokine production. Uptake of [3H]thymidine by mouse CD4 T cells stimulated with anti-CD3 mAbs plus either anti-CD28 or IL-7 was inhibited up to 50% by 10−9–10−6 M S1P. Suppression by S1P required Ca2+ signaling and was reduced by intracellular cAMP. S1P decreased CD4 T cell generation of IFN-γ and IL-4, without affecting IL-2. A Th1 line from D011.10 TCR transgenic mice without detectable S1P1 was refractory to S1P until introduction of S1P1 by retroviral transduction. S1P then evoked chemotaxis, inhibited chemotaxis to CCL-5 and CCL-21, and suppressed Ag-stimulated proliferation and IFN-γ production. Thus, S1P1 signals multiple immune functions of T cells as well as migration and tissue distribution.

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC 1R and VPAC2R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC1R is expressed constitutively, whereas VPAC2R is induced by immune stimuli. VPAC2R-null (VPAC2R ؊/؊ ) mice on a C57BL͞6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3 ؉ ) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayedtype hypersensitivity (DTH) was significantly enhanced in VPAC2R-null mice compared with age-and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were >70% lower in VPAC2R-null mice than in wild-type controls. Cytokine production by splenic CD4 ؉ T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean ‫؍‬ 3-fold) and IFN-␥ (mean ‫؍‬ 3-fold), and lower levels of IL-4 (mean ‫؍‬ one-fifth) in VPAC2R-null mice than wild-type controls. Loss of VIP-VPAC2R maintenance of the normal ratio of Th2͞Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immunemediated diseases.is produced by cholinergic and sensory nerves, including those in thymus, spleen, and lymph nodes, and by T cells (1-3). VIP has potent effects on T cell differentiation, migration, and generation of diverse cytokines (4-12). Production of some cytokines by the two subsets of mouse helper T (Th) cells in vitro is regulated differentially by VIP. VIP inhibits release of IL-2 from mouse type 1 Th (Th1) cells, which mediate classical delayed-type cellular immunity, and from type 2 Th (Th2) cells and enhances release of IL-5 from mouse Th2 cells, which mediate acute and subacute hypersensitivity reactions, such as allergy. Effects of VIP on the generation of many other cytokines by Th cells, however, is variably dependent on the source and state of activation of the Th cells. Type I G protein-coupled VIP receptors (VPAC 1 Rs) are highly expressed constitutively by unstimulated Th cells in mouse blood and spleen, whereas the homologous VPAC 2 Rs are expressed at low levels or absent (13-18). However, VPAC 2 Rs are upregulated to high levels and VPAC 1 Rs down-regulated by Th cell stimulation, suggesting that VPAC 2 Rs are the dominant transducer of effects of VIP on activated Th cells (19)(20)(21)(22). Investigations of the capacity of VIP to suppress Th1-mediated delayedtype hypersensitivity (DTH) and to enhance Th2-dependent immediate-type hypersensitivity reactions in vivo through VPAC 2 Rs have been hampered by the lack of effective pharmacological agents. A transgenic (TG) mouse model has been developed in which normally inducible VPAC 2 Rs are constitutively expressed in CD4 ϩ (helper-inducer) T cells of the Th1 cell-dominant C57BL͞6 strain of mice, at levels similar t...

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and macrophages mediate T cell functions. Endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) are specific for S1P (Edg-1, -3, -5, and -8 Rs) and LPA (Edg-2, -4, and -7 Rs). Human T cell tumors express many Edg Rs for both LPA and S1P. In contrast, human blood CD4+ T cells express predominantly Edg-4, and CD8+ T cells show only traces of Edg-2 and -5, by quantification of mRNA and Edg R Ags. LPA at 10−10–10−6 M suppressed significantly the secretion of IL-2 from anti-CD3 plus anti-CD28 Ab-challenged CD4+ T cells, but not CD8+ T cells. Monoclonal anti-Edg-4 R Ab, like LPA, suppressed stimulated IL-2 secretion from CD4+ T cells, but not CD8+ T cells. Constitutive expression of Edg-4 by CD4+, but not CD8+, human T cells accounts for differential functional responsiveness of the T cell subsets to LPA.