A general property of signal transduction pathways is that prolonged stimulation decreases responsiveness, a phenomenon termed desensitization. Yeast cells stimulated with mating pheromone activate a heterotrimeric G-protein-linked, MAP-kinase-dependent signalling pathway that induces G1-phase cell-cycle arrest and morphological differentiation (reviewed in refs 1, 2). Eventually the cells desensitize to pheromone and resume growth. Genetic studies have demonstrated the relative importance of a desensitization mechanism that uses the SST2 gene product, Sst2p. Here we identify a mammalian gene family termed RGS (for regulator of G-protein signalling) that encodes structural and functional homologues of Sst2p. Introduction of RGS family members into yeast blunts signal transduction through the pheromone-response pathway. Like SST2 (refs 8-10), they negatively regulate this pathway at a point upstream or at the level of the G protein. The RGS family members also markedly impair MAP kinase activation by mammalian G-protein-linked receptors, indicating the existence and importance of an SST2-like desensitization mechanism in mammalian cells.

Using bone marrow derived mast cells from SH2-containing inositol-5-phosphatase (SHIP) ؉/؉ and ؊/؊ mice, we found that the loss of SHIP leads to a dramatic increase in Steel Factor (SF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ), a substantial reduction in PI(3,4)P 2 , and no change in PI(4,5)P 2 levels. We also found that SF-induced activation of protein kinase B (PKB) is increased and prolonged in SHIP؊/؊ cells, due in large part to more PKB associating with the plasma membrane in these cells. Pretreatment of SHIP؊/؊ cells with 25 M LY294002 resulted in complete inhibition of SFinduced PI(3,4)P 2 , while still yielding PI(3,4,5)P 3 levels similar to those achieved in SHIP؉/؉ cells. This offered a unique opportunity to study the regulation of PKB by PI(3,4,5)P 3 , in the absence of PI(3,4)P 2 . Under these conditions, PKB activity was markedly reduced compared with that in SF-stimulated SHIP؉/؉ cells, even though more PKB localized to the plasma membrane. Although phosphoinositide-dependent kinase 1 mediated phosphorylation of PKB at Thr-308 was unaffected by LY294002, phosphorylation at Ser-473 was dramatically reduced. Moreover, intracellular delivery of PI(3,4)P 2 to LY294002-pretreated, SF-stimulated SHIP؊/؊ cells increased phosphorylation of PKB at Ser-473 and increased PKB activity. These results are consistent with a model in which SHIP serves as a regulator of both activity and subcellular localization of PKB.The src homology 2 (SH2 1 )-containing inositol phosphatase (SHIP) is a 145-kDa hemopoietic-specific signaling protein (1-3) that becomes both tyrosine-phosphorylated and associated with the adapter protein Shc in response to many cytokines and to B and T cell receptor engagement (4). SHIP has been shown to inhibit immune receptor activation in both mast cells and B cells by binding to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibition motif of the inhibitory co-receptor Fc␥RIIB and inhibiting Fc⑀R1-and B cell receptor-induced calcium influx, respectively (5, 6). In addition, SHIP has been shown, even in the absence of Fc␥RIIB coclustering, to play a "gatekeeper" role in IgE-mediated mast cell degranulation by setting the threshold for and limiting the degranulation process (7,8).In 1996 when we and others first reported the cloning of SHIP (1-3), we demonstrated its ability, in vitro, to hydrolyze the 5Ј-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) but not from PI(4,5)P 2 . More recently, however, by modifying the in vitro assay conditions, SHIP was found capable of readily hydrolyzing PI(4,5)P 2 to PI(4)P (9, 10). To resolve its phospholipid substrate specificity and to gain some insight into the normal role that SHIP plays in vivo, we generated a SHIP knockout mouse by homologous recombination in embryonic stem cells (11). Although these mice are viable and fertile, they suffer from progressive splenomegaly, massive myeloid infiltration of the lungs, wasting, and a shortened lifespan (11,12). Interestingly, granulocyte/macroph...

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-α. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various Gα subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Giα and Gqα GTPase-activating protein and a potential G12α effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12α. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.