Behavioural immune landscapes of inflammation.

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

Functions and mechanisms of the GPCR adaptor protein Norbin.

Norbin (Neurochondrin, NCDN) is a highly conserved 79 kDa adaptor protein that was first identified more than a quarter of a century ago as a gene up-regulated in rat hippocampus upon induction of long-term potentiation. Most research has focussed on the role of Norbin in the nervous system, where the protein is highly expressed. Norbin regulates neuronal morphology and synaptic plasticity, and is essential for normal brain development and homeostasis. Dysregulation of Norbin is linked to a variety of neurological conditions. Recently, Norbin was shown to be expressed in myeloid cells as well as neurons. Myeloid-cell specific deletion revealed an important role of Norbin as a suppressor of neutrophil-derived innate immunity. Norbin limits the ability of neutrophils to clear bacterial infections by curbing the responsiveness of these cells to inflammatory and infectious stimuli. Mechanistically, Norbin regulates cell responses through binding to its interactors, in particular to a wide range of G protein-coupled receptors (GPCRs). Norbin association with GPCRs controls GPCR trafficking and signalling. Other important Norbin interactors are the Rac guanine-nucleotide exchange factor P-Rex1 and protein kinase A. Downstream signalling pathways regulated by Norbin include ERK, Ca2+ and the small GTPase Rac. Here, we review the current understanding of Norbin structure, expression and its roles in health and disease. We also explore Norbin signalling through its interactors, with a particular focus on GPCR trafficking and signalling. Finally, we discuss avenues that could be pursued in the future to increase our understanding of Norbin biology.

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis.

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of PREX1 mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher PREX1 mRNA in ER+ve/luminal tumors was associated with poor outcome in luminal B cancers. Prex1 deletion in MMTV-neu or MMTV-PyMT mice reduced Rac1 activation in vivo and improved survival. High level MMTV-driven transgenic PREX1 expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV-PREX1 expression in MMTV-neu mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.

Rac-GTPases and their Rac-GEF activators play important roles in the recruitment and host defence functions of neutrophils. These proteins control the activation of adhesion molecules and the cytoskeletal dynamics that enable the adhesion, migration and tissue recruitment of neutrophils. They also regulate the effector functions that allow neutrophils to kill bacterial and fungal pathogens, and to clear debris. This review focuses on the roles of Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.

Platelets in neutrophil recruitment to sites of inflammation.

PURPOSE OF REVIEW: This review describes the essential roles of platelets in neutrophil recruitment from the bloodstream into inflamed and infected tissues, with a focus on recent findings. RECENT FINDINGS: Platelets are required for the recruitment of neutrophils to sites of inflammation and infection. They fulfil this role largely by enabling contacts of circulating neutrophils with the inflamed blood vessel wall prior to extravasation. Platelets promote both early stages of neutrophil recruitment (tethering, rolling, arrest, firm adhesion) and - as recent work has demonstrated - later stages (intravascular crawling and diapedesis). Recent studies have also begun to identify platelet-signaling pathways that can elicit the underlying interactions between platelets, neutrophils and vascular endothelial cells without stimulating concomitant platelet aggregation and thrombus formation. These pathways include Rho-guanine-nucleotide binding proteins and Rho-guanine-nucleotide exchange factors. SUMMARY: Recent findings have contributed to our burgeoning understanding of the platelet-dependent mechanisms that control neutrophil recruitment to sites of inflammation and have opened up new avenues of research aimed at increasing our knowledge of these mechanisms further. These insights might lead to the development of novel anti-inflammatory drugs that will be useful in a wide range of inflammatory diseases without causing immunodeficiency.

Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gßγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gßγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1.

P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of inflammation.

The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1(-/-) Vav1(-/-) (P1V1) or P-Rex1(-/-) Vav3(-/-) (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L- and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a platelet-dependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment.

Small GTPases and their guanine-nucleotide exchange factors and GTPase-activating proteins in neutrophil recruitment.

PURPOSE OF REVIEW: The review describes the roles of Rho- and Rap-guanosine triphosphatases (GTPases) and of their activators, guanine-nucleotide exchange factors (GEFs), and inhibitors, GTPase activating proteins (GAPs), in neutrophil recruitment from the blood stream into inflamed tissues, with a focus on recently identified roles in neutrophils, endothelial cells, and platelets. RECENT FINDINGS: Recent studies have identified important roles of Rho- and Rap-GTPases, and of their GEFs and GAPs, in the neutrophil recruitment cascade. These proteins control the upregulation and/or activation of adhesion molecules on the surface of neutrophils, endothelial cells, and platelets, and they alter cell/cell adhesion in the vascular endothelium. This enables the capture of neutrophils from the blood stream, their migration along and through the vessel wall, and their passage into the inflamed tissue. In particular, it has recently become clear that P-Rex and Vav family Rac-GEFs in platelets are crucial for neutrophil recruitment. SUMMARY: These recent findings have contributed greatly to our understanding of the signalling pathways that control neutrophil recruitment to sites of inflammation and have opened up new avenues of research in this field.

P-Rex1 directly activates RhoG to regulate GPCR-driven Rac signalling and actin polarity in neutrophils.

G-protein-coupled receptors (GPCRs) regulate the organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine-nucleotide-exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and phosphoinositide 3-kinase (PI3K) in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils. Furthermore, loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and Rac-dependent NADPH oxidase activity, suggesting they both function upstream of Rac in this system. Loss of RhoG also impaired GPCR-driven recruitment of the Rac GEF DOCK2, and F-actin, to the leading edge of migrating neutrophils. Taken together, our results reveal a new signalling hierarchy in which P-Rex1, acting as a GEF for RhoG, regulates Rac-dependent functions indirectly through RhoG-dependent recruitment of DOCK2. These findings thus have broad implications for our understanding of GPCR signalling to Rho GTPases and the actin cytoskeleton.

P-Rex1 and Vav1 cooperate in the regulation of formyl-methionyl-leucyl-phenylalanine-dependent neutrophil responses.

G protein-coupled receptor (GPCR) activation elicits neutrophil responses such as chemotaxis and reactive oxygen species (ROS) formation, which depend on the small G protein Rac and are essential for host defense. P-Rex and Vav are two families of guanine-nucleotide exchange factors (GEFs) for Rac, which are activated through distinct mechanisms but can both control GPCR-dependent neutrophil responses. It is currently unknown whether they play specific roles or whether they can compensate for each other in controlling these responses. In this study, we have assessed the function of neutrophils from mice deficient in P-Rex and/or Vav family GEFs. We found that both the P-Rex and the Vav family are important for LPS priming of ROS formation, whereas particle-induced ROS responses and cell spreading are controlled by the Vav family alone. Surprisingly, fMLF-stimulated ROS formation, adhesion, and chemotaxis were synergistically controlled by P-Rex1 and Vav1. These responses were more severely impaired in neutrophils lacking both P-Rex1 and Vav1 than those lacking the entire P-Rex family, the entire Vav family, or both P-Rex1 and Vav3. P-Rex1/Vav1 (P1V1) double-deficient cells also showed the strongest reduction in fMLF-stimulated activation of Rac1 and Rac2. This reduction in Rac activity may be sufficient to cause the defects observed in fMLF-stimulated P1V1 neutrophil responses. Additionally, Mac-1 surface expression was reduced in P1V1 cells, which might contribute further to defects in responses involving integrins, such as GPCR-stimulated adhesion and chemotaxis. We conclude that P-Rex1 and Vav1 together are the major fMLFR-dependent Dbl family Rac-GEFs in neutrophils and cooperate in the control of fMLF-stimulated neutrophil responses.

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gßγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gßγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1ß, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis.

Introduction: Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Methods: Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. Results: All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Discussion: Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

The GPCR adaptor protein Norbin regulates S1PR1 trafficking and the morphology, cell cycle and survival of PC12 cells.

Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking.

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating ß2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and ß2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities.

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.

Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses.

Host defense against bacterial and fungal infections diminishes with age. In humans, impaired neutrophil responses are thought to contribute to this decline. However, it remains unclear whether neutrophil responses are also impaired in old mice. Here, we investigated neutrophil function in old mice, focusing on responses primed by lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria like E. coli, which signals through toll-like receptor (TLR) 4. We show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment was elevated during LPS-induced but not aseptic peritonitis. Neutrophils from old mice showed reduced killing of E. coli. Their reactive oxygen species (ROS) production was impaired upon priming with LPS but not with GM-CSF/TNFα. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas impairment of NET release in response to S. aureus was independent of LPS. Unexpectedly, chemotaxis was normal, as were Rac1 and Rac2 GTPase activities. LPS-primed activation of Erk and p38 Mapk was defective. PIP3 production was reduced upon priming with LPS but not with GM-CSF/TNFα, whereas PIP2 levels were constitutively low. The expression of 5% of neutrophil proteins was dysregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as TLR-pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. The upregulation of CD180 and downregulation of MyD88 likely contribute to the impaired LPS signaling. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS/TLR4 pathway dependence resolves previous controversy regarding effects of age on murine neutrophils and confirms that mice are an appropriate model for the decline in human neutrophil function.

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination.

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2(-/-) pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2(-/-) and P-Rex1(-/-)/P-Rex2(-/-) mice. P-Rex2(-/-) mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1(-/-)/P-Rex2(-/-) mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

Rho Family GTPases and Rho GEFs in Glucose Homeostasis.

Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic ß cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and ß-PIX were found to control the glucose-stimulated release of insulin by pancreatic ß cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis.

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation.