title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.
abstract: 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 Gβγ 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 Gβγ-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.
authors.affiliation: From the Signalling Programme and.
authors.forename: Dingxin
authors.initials: D
authors.lastname: Pan
authors.affiliation: From the Signalling Programme and.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Barber
authors.affiliation: From the Signalling Programme and.
authors.forename: Kirsti
authors.initials: K
authors.lastname: Hornigold
authors.affiliation: From the Signalling Programme and.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baker
authors.affiliation: From the Signalling Programme and.
authors.forename: Judit M
authors.initials: JM
authors.lastname: Toth
authors.affiliation: the Mass Spectrometry Facility, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Oxley
authors.affiliation: From the Signalling Programme and heidi.welch@babraham.ac.uk.
authors.forename: Heidi C E
authors.initials: HC
authors.lastname: Welch
mesh_terms: D000818:Animals; D001921:Brain; D019556:COS Cells; D048430:Cell Shape; D022081:Cell Surface Extensions / Q000378:metabolism; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000502:physiology*; D009928:Organ Specificity; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; C511197:PREX1 protein, mouse; C422102:SFAP75 protein, mouse
keywords: G protein-coupled receptor (GPCR); Neurochondrin (NCDN); PREX1; PREX2; Rac (Rac GTPase); Rho (Rho GTPase); cell signaling; guanine nucleotide exchange factor (GEF); protein complex; small GTPase