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

The Journal of Biological Chemistry
Dingxin PanHeidi C E Welch

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 ...Continue Reading

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Citations

Sep 3, 2016·Small GTPases·Marcelo G KazanietzCynthia Lopez-Haber
Apr 24, 2018·European Journal of Clinical Investigation·Chiara Pantarelli, Heidi C E Welch
Dec 12, 2018·The Journal of Biological Chemistry·Sendi Rafael Adame-GarcíaJosé Vázquez-Prado
Jul 16, 2017·Biochemical Society Transactions·Nuthasuda SrijakotreChristina A Mitchell
Aug 2, 2018·Biochemical Society Transactions·Victoria Casado-MedranoMarcelo G Kazanietz

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Methods Mentioned

BETA
nucleotide
GTPases
GTPase
scraping
Pulldown
coimmunoprecipitation
immunoprecipitation

Software Mentioned

Nikon Elements
ImageJ

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