Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.

The monomeric GTPase Rac and the lipid kinase phosphoinositide 3-kinase (PI3K) are intracellular signalling enzymes that each regulate a huge range of cellular functions. Their signalling pathways overlap. Several pathways lead from PI3K activation via the production of the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P 3 ) to the activation of guanine-nucleotide exchange factors (GEFs) that activate Rac. Vice versa, Rac can also stimulate the activation of PI3K, although the mechanism for this is unclear. We review here the evidence that links PI3K and Rac signalling pathways.

Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) and the G␤␥ subunits of heterotrimeric G proteins. We show that a PRex1 mutant lacking the PH domain (⌬PH) cannot be stimulated by PtdIns(3,4,5)P 3 , which implies that the PH domain confers PtdIns(3,4,5)P 3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P 3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P 3 -stimulated P-Rex1 activity. The Rac-GEF activities of the ⌬PH mutant and the DH/PH domain tandem can both be stimulated by G␤␥ subunits, which infers that G␤␥ subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P 3 or G␤␥ subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.The P-Rex family (P-Rex1, P-Rex2, and P-Rex2B) are guanine-nucleotide exchange factors (GEFs) 1 (1-3), enzymes that catalyze the dissociation of GDP from small GTPases, allowing excess free GTP to bind, and thus rendering the GTPases active. The substrate of the P-Rex family is the Rho family GTPase Rac (1-3), an enzyme that regulates a wide range of cell functions, including cell shape, movement, secretion, phagocytosis, transcription, translation, and the production of reactive oxygen species. Depletion of P-Rex1 from neutrophil-related cell lines results in low reactive oxygen species formation via G protein-coupled receptors (1).More than a dozen Rac-GEFs are known apart from the P-Rex family, for example, the Vav, Sos, and Tiam families (4). Their Rac-GEF activities are tightly regulated, often by a combination of mechanisms. Some are activated by phosphorylation through protein kinases, e.g. Vav1 and Ras-GRF1 by Src family protein tyrosine kinases (5, 6), Sos1 by Abl (7), and Tiam1 by Ca 2ϩ /calmodulin-dependent protein kinase II (8). Another activating mechanism is binding to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ), a lipid second messenger produced by class I phosphoinositide 3-kinase. PtdIns(3,4,5)P 3 stimulates the Rac-GEF activities of P-Rex1, SWAP-70, Vav1, and Sos1 (9). Finally, Rac-GEFs can be activated by protein interactions. For example, Tiam1 is activated by binding to GTP-Ras (10), Sos1 by the protein complex Abi1-Eps8 (11), and P-Rex family enzymes by the ␤␥ subunits of heterotrimeric G proteins (1, 2).The basic element for Rac-GEF activity is the catalytic Dbl homology (DH) domain. Regulating accessibi...

The T-cell immune response is directed against antigenic peptide fragments generated in intracellular compartments, the cytosol or the endocytic system. Peptides derived from cytosolic proteins, usually of biosynthetic origin, are presented efficiently to T-cell receptors by major histocompatibility complex (MHC) class I molecules, with which they assemble, probably in the endoplasmic reticulum (ER). In the absence of recognizable N-terminal signal sequences, such cytosolic peptides must be translocated across the ER membrane by a novel mechanism. Genes apparently involved in the normal assembly and transport of class I molecules may themselves be encoded in the MHC. Here we show that one of these, the rat cim gene, maps to a highly polymorphic part of the MHC class II region encoding two novel members of the family of transmembrane transporters related to multidrug resistance. Other members of this family of transporter proteins are known to be capable of transporting proteins and peptides across membranes independently of the classical secretory pathway. Such molecules are credible candidates for peptide pumps that move fragments of antigenic proteins from the cytosol into the ER.