Upon food intake, the glucose level in circulating blood increases by trans-cellular transport of glucose from the intestinal lumen into the blood. However, the insulin secreted from the pancreatic β-cells directs the individual cells to absorb glucose for energy source to result in the maintenance of the normal blood glucose level. Elevated insulin in blood is sensed by the insulin receptor of the individual cells, and the propagation of the insulin signaling pathway drives the glucose uptake. The final cellular glucose uptake from the blood into the cells is performed by trafficking of vesicles containing the glucose transporter protein (e.g. GLUT4) from the intracellular pools to the cell surfaces. As in most of the vesicle budding, uncoating, docking and fusion processes, this translocation of GLUT4 vesicles are tightly regulated by Rab·GTPases (Rabs) and Rab·GTPase-activating proteins (RabGAPs). Rabs are GTPases with intrinsic activity, however RabGAPs enhance the low hydrolysis rate of Rab-bound GTP. The two major cell types that absorb glucose, which are the adipocytes and the skeletal myocytes, respectively encode TBC1D4 (also known as AS160) and TBC1D1 which are integral RabGAPs in the insulin-mediated GLUT4 vesicle trafficking event. TBC1D4 and TBC1D1 are multi-domain RabGAP proteins which consist of two putative N-termini phosphotyrosine binding (PTB) domains and a C-terminal catalytic RabGAP domain that are linked by regions containing several Akt(PKB)-mediating serine and threonine phosphorylation sites. In the basal state, TBC1D4 (or TBC1D1)-catalyzed hydrolysis of Rab-bound GTP (active) to GDP (inactive) leaves GLUT4 vesicles sequestered in an intracellular compartment. However, insulin signal results in the activation of Akt (PKB) and the consequent phosphorylation of TBC1D4 (or TBC1D1). The Akt phosphorylation of TBC1D4 (or TBC1D1) deactivates the Rab·GTP hydrolysis activity by an unknown mechanism, and concomitantly results in the increase of Rab·GTP concentrations. The active Rab·GTP in turn promotes GLUT4 vesicle translocation to the cell membrane and finally stimulates the glucose uptake from blood into the cells. X-ray crystal structures of the human TBC1D1 RabGAP domain and TBC1D4 RabGAP domain (PDB code: 3QYE & 3QYB, respectively) have been recently reported. Since, TBC1D1 and TBC1D4 share 76% sequence identity over their RabGAP domains, the secondary structure elements mostly overlapped in the two structures which were predominately α-helical with no β-sheet elements. The 17 αhelices in TBC1D1 RabGAP domain were sequentially named from α1 to α16 ending with α16'. However, the oligomeric association states of the two TBC1D1 and TBC1D4 RabGAP domains in the crystal asymmetric unit were different resulting from the presence (or absence) of a single helix α16'. TBC1D1 RabGAP domain containing the C-terminal α16' forms an asymmetric dimer with α16' of one molecule interacting with α16 of the other molecule (Figure 1). However in TBC1D4 RabGAP domain, α16' in the protein construct had to be deleted for crystallization and enhanced X-ray diffraction (Figure 2), and this results in TBC1D4 RabGAP domain being a monomer in the crystal (Figure 1). Certain is the fact that α16' mediates the resulting differences in the oligomeric states, the biological relevance of this asymmetric dimer cannot be concluded only from the association modes within the crystal. Since only two αhelices (α16' and α16) of TBC1D1 mediate the dimerization, it was suspected that the dimer is a result of crystal packing artifacts, and is non-relevant outside the context of the crystal. However in this study, we have compared the oligomeric association states of TBC1D1 and TBC1D4 RabGAP domains (Figure 2) using analytical ultracentrifugation (AUC) techniques to address the state of interaction in solution. Sedimentation equilibrium experiments were performed in a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge using 12 mm carbon-filled epoxy double-sector centerpieces and quartz windows at optical density of 0.25 at 280 nm (6.67 μM and 6.69 μM for TBC1D1 and TBC1D4 RabGAP domains, respectively). The measurements were performed at two different rotor speeds of 13,000 and 18,000 RPMs at 20 C. The proteins were in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 1.5 mM TCEP. The sedimentation equilibrium data were analyzed using MLAB software (Civilized Software, Inc., Silver Spring, MD) using the following equation: C(r) = cb*exp (Ap* Mp* (r − rb) + e (1)
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Jan 1, 2013
A I Mis'Kevich + 2
Open Access