title: Analysis of connecdenn 1-3 (DENN1A-C) GEF activity for Rab35.
abstract: Rabs (Ras-related proteins in brain) form the largest family of small GTPases and control numerous aspects of membrane trafficking at multiple cellular sites. Rab GTPases toggle between an inactive GDP-bound state and an active GTP-bound state. Activation of Rab GTPases requires guanine nucleotide exchange factors (GEFs) that interact with inactive GDP-bound Rabs and catalyze the removal of GDP, allowing GTP to bind. The largest single family of GEFs for Rabs is comprised of proteins bearing a DENN (differentially expressed in normal and neoplastic cells) domain. In this chapter we describe a biochemical method that directly measures the exchange activity of DENN domains by monitoring loading of GTP onto a Rab GTPase. Rabs are first purified from bacterial or mammalian sources and are then loaded with GDP. Purified DENN domains or DENN domain-bearing proteins are added in the presence of [(35)S]GTPγS and the transfer of [(35)S]GTPγS to the Rab is measured by filtering the reaction over nitrocellulose membranes to trap the Rab and thus the associated [(35)S]GTPγS.
authors: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.
authors: Patrick D
authors: PD
authors: Allaire
authors: Peter S
authors: PS
authors: McPherson
authors: Brigitte
authors: B
authors: Ritter
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C511183:RAB35 protein, human; D020691:rab GTP-Binding Proteins