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Overview
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Product Uses Include:
Analysis of in vivo Arf6 activation.
Introduction
The mammalian ADP-ribosylation factor (Arf) subfamily of Ras-related small G-proteins was originally named for the ability to stimulate cholera toxin mediated ADP-ribosylation of the Gsα subunit utilized by many GPCRs (1). The Arf GTPases have been grouped into three classes based on their size and amino acid similarity (2): class I (Arf6 and Arf3), class II (Arf4 and Arf5) and class III (Arf6). One of the primary roles of Arf6 is in protein trafficking from the plasma membrane to endocytic compartments (e.g., GPCRs) and appears to have a role in the anterograde transport of some GPCRs (3, 4). Like Arf1, Arf6 functions in part by the activation of lipid modifying enzymes that alter the local membrane environment (3).
Arf6, like other small G-proteins, cycles between the inactive GDP-bound and the active GTP-bound states. The preferential association of effector proteins with the GTP-bound over the GDP-bound state of Arf6 provides the basis for Arf6’s function in the cell. This highly specific association of effector proteins with Arf6-GTP has been exploited to develop affinity precipitation assays to monitor Arf6 activation (5).
Our Arf6 Activation Assay Biochem Kit™ utilizes the Arf6 protein binding domain (PBD) of the effector protein GGA3 (Golgi-localized γ-ear containing, Arf-binding protein 3), which has been shown to specifically bind the GTP-bound form of Arf6 (5, 6). We have covalently conjugated purified GGA3-PBD (amino acids 1-316) expressed in E. coli to the colored sepharose beads provided in this kit. Using these beads, the researcher is able to “pull-down” Arf6-GTP and quantify the level of active Arf6 with a subsequent Western blotting step using the Arf6 specific antibody provided in this kit. This assay provides a simple means of analyzing cellular Arf6 activation levels in a variety of systems.
References
Kahn, R. A., and Gilman, A. G. (1986) The Protein Cofactor for ADP-ribosylation of Gs by Cholera Toxin is Itself a GTP Binding Protein.J. Biol. Chem.261, 7906-7911.
Tsuchiya, M., Price, S. R., Tsai, S-C, Moss, J. and Vaughan, M. (1991) Molecular Identification of ADP-Ribosylation Factor mRNAs and Their Expression in Mammalian Cells.J. Biol. Chem.266, 2772-2777.
D’Souza-Schorey, C., and Chavrier, P. (2006) ARF proteins: Roles in membrane traffic and beyond.Nat. Rev. Mol. Cell Biol.7, 347-358.
Wang, G., and Wu, G. (2012) Small GTPase regulation of GPCR anterograde trafficking.Trends Pharmacol. Sci.33, 28-34.
Cohen, L. A., and Donaldson, J. G. (2010) Analysis of Arf GTP-binding protein function in cells.Curr. Protoc. Cell Biol.48, 14.12.1-14.12.17.
Takatsu, H., Yoshino, K., Toda, K., and Nakayama, K. (2002) GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADPribosylation factors. Biochem. J. 365, 369-378.
Balasubramanian, N., Scott, D. W., Castle, J. D., Casanova, J. E., and Schwartz, M. A. (2007) Arf6 and microtubules in adhesion-dependent trafficking of lipid rafts.Nat. Cell Biol. 9, 1381-1391.
Goldfinger, L. E., Ptak, C., Jeffery, E. D., Shabanowitz, J., Hunt, D. F., and Ginsberg, M. H. (2006) RLIP76(RalBP1) is an R-Ras effector that mediates adhesiondependent Rac activation and cell migration.J. Cell Biol.174, 877-888.Please contact us at for specific academic pricing.
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Overview