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N.A. Finch, P.J. Linser, J.D. Ochrietor; A Study of the Interaction Between the Basigin Transmembrane Domain and MCT1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3818.
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It has been demonstrated that monocarboxylate transporter–1 (MCT1) interacts with Basigin using immunoprecipitation and fluorescence resonance energy transfer (FRET) techniques. It has been hypothesized that these two membrane proteins interact via the transmembrane domain of Basigin and that the glutamate residue within the otherwise hydrophobic domain is necessary for this interaction. We therefore sought to determine whether the transmembrane region of Basigin does indeed interact with MCT1 and also whether the glutamate residue is required.
A full–length 6X–histidine–tagged transmembrane protein (24 amino acids), as well as 6X–histidine–tagged deletion mutants were generated using the pET102/D vector (Invitrogen). The domain was truncated from both the N– and C–termini at intervals of six amino acids. These proteins were used for Far Western blotting analyses. Mouse retina proteins were separated via SDS–PAGE and transferred to a nitrocellulose membrane. The membrane was then probed with a 6X–histidine–tagged protein, followed by an anti–6X–histidine antibody, and the appropriate alkaline phosphatase–tagged secondary antibody.
We found that the full–length transmembrane domain bound to a retina protein of ∼45 kDa, which is the mass of MCT1. When only the first six amino acids of the domain were present, complete loss of binding was observed. Proteins from the central portion of the domain, which contain the glutamate residue, showed only faint binding. However, when only the last six amino acids of the domain were present, a binding pattern similar to that of the full–length domain was observed.
These data suggest that the glutamate residue within the Basigin transmembrane domain is not necessary for the interaction with MCT1. The results also indicate that the last six amino acids of the domain are important for binding to MCT1. Future studies will be aimed at confirming that MCT1 is the 45 kDa binding protein in mouse retina lysates. Also, site directed mutagenesis will be performed to study the contribution of each of the last six amino acids to this binding interaction.
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