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

Abstract

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.

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

“…PC12 rat phaeochromocytoma cells were a kind gift from Dr Llewelyn Roderick, Babraham Institute, Cambridge, UK. From these, we derived PC12-S1PR1 cells which stably express C-terminally GFP-tagged S1PR1, as described previously 28 . Cells were maintained in complete Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, 41,965–039), supplemented with 10% horse serum, 5% foetal bovine serum, 1 U/ml penicillin and 1 U/ml streptomycin (Gibco, 15,140–122), 2 mM glutamine, and 500 µg/ml G-418 disulphate (Melford, Ipswich, UK, G0175)) in poly-D-lysine coated vented tissue culture flasks (Nunc) at 37 °C in a humidified, 5% CO 2 incubator.…”

“…PC12-S1PR1 cells were transfected with this sgRNA and with a GeneArt CRISPR Nuclease Vector that expresses orange fluorescent protein (OFP) (Thermo Fisher Scientific, A21174) using JetPEI transfection reagent (Polyplus 101-10N), following the manufacturer’s protocol. 44 h later, the cells were resuspended in DMEM, 1% FBS, 2 mM glutamine, and OFP-positive cells were enriched by fluorescence-activated cell sorting as described 28 . Single cells were sorted into 96-well plates and cultured for clonal expansion in the complete growth medium.…”

“…Cell growth, viability, and cell cycle stages were assessed essentially as described 28 . Briefly, wild type and Norbin-deficient PC12-S1PR1 cells were seeded into poly-D-lysine coated 12-well tissue culture plates (Nunc, 150628) at a density of 2 × 10 4 cells per well.…”

“…They express several neuronal receptors, are capable of neurotransmitter secretion, morphologically resemble neurons upon differentiation with nerve-growth factor (NGF), and are amenable to experimental manipulation 27 . PC12-S1PR1 is a PC12 cell line that we recently established, which stably expresses S1PR1 with a C-terminal GFP tag, at a level approximately 3% over the abundant endogenous S1PR1 28 . Here, we generated Norbin-deficient PC12-S1PR1 cells by CRISPR/Cas9-mediated knockout to compare cell responses, as well as S1PR1 trafficking and signalling, between wild type and Norbin-deficient PC12-S1PR1 cells.…”

At the Research Frontiers of Small GTPases

“…Kristensson reviewed the GTP-binding domains of α-, β-, and γ-tubulin and elaborated on the functions of the γ-tubulin GTP-binding domain in the regulation of the γ-tubulin meshwork dynamics and cellular homeostasis [ 5 ]. In the only original research paper in the collection, Hampson and colleagues revealed the role of P-Rex1, a GEF that activates Rac small GTPases in response to G-protein-coupled receptors (GPCR) activation, in sphingosine 1-phosphate (S1P) signaling [ 6 ]. The authors showed, using PC12 cells that stably overexpressed the S1P receptor, that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt.…”