title: In Vitro Assay for the Rap GTPase-Activating Protein Activity of the Purified Cytoplasmic Domain of Plexin.
abstract: Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, 75390-9041, USA.
authors: Heath G
authors: HG
authors: Pascoe
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, 75390-9041, USA.
authors: Yuxiao
authors: Y
authors: Wang
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, 75390-9041, USA. Xuewu.zhang@utsouthwestern.edu.|Department of Biophysics, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX, 75390-9041, USA. Xuewu.zhang@utsouthwestern.edu.
authors: Xuewu
authors: X
authors: Zhang
mesh_terms: D015815:Cell Adhesion Molecules / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; D009419:Nerve Tissue Proteins; C091529:plexin; D020558:GTP Phosphohydrolases; C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins
keywords: GAP; GAP activity assay; Plexin; Rap; Semaphorin