CD9 Tetraspanin Interacts with CD36 on the Surface of Macrophages: A Possible Regulatory Influence on Uptake of Oxidized Low Density Lipoprotein

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL

Sample Preparation and Technical Setup for NMR Spectroscopy with Integral Membrane Proteins

NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the α-helical membrane protein MsbA and the β-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueous solution. Since MsbA and BamA are typical examples of their respective architectural classes, the protocols presented here can also serve as a reference for other integral membrane proteins