No abstract

The consequences of the binding of annexin V on the structure and dynamics of PC/PS bilayers were studied by means of fluorescence polarization, 31P NMR, 2H NMR, and fluorescence recovery after photobleaching (FRAP). Even at complete coverage of the lipid bilayers by the protein, annexin V showed no influence on the lipid molecular packing and the acyl chain flexibility of both PC and PS. The fluorescence polarization of the probe DPH, the 31P NMR spectra, and deuterium quadrupolar splittings of P(d31)OPS remained unchanged. However, upon binding of annexin V, two distinct populations of PC were visible in 2H NMR, which were in slow exchange on the deuterium NMR time scale (microseconds). One component in the spectrum was identical to the protein-free sample, while a second, broad, component appeared. The presence of the protein induced a decrease in the transverse relaxation times (T2e), indicative of the appearance of slow motions (milliseconds to microseconds), in the P(d31)-OPS spectrum and in the P(d31)OPC broad component. FRAP experiments were carried out with the probes C12-NBD-PC and C12-NBD-PS: at saturation, annexin V reduced the lateral diffusion rate of PC by 40% and nearly blocked the diffusion of PS. These combined experiments are consistent with a model in which annexin V enters a proteolipidic complex in the form of an extended 2D network, stabilized by specific interactions with PS. As seen from the lateral diffusion rates and the acyl chains NMR spectral parameters, two separate lipid populations appear, presumably corresponding to those interacting with annexinV (PC and PS) and protein free domains (mainly PC).

This study provides evidence that the differences in membrane composition found from one cell type to another can represent a limiting factor to recovering the functionality of transmembrane proteins when expressed in heterologous systems. Restoring the properties of the human -opioid receptor in yeast (Saccharomyces cerevisiae), similar to those observed in native cells, was achieved by replacing ergosterol from yeast by cholesterol, which is normally found in mammalian plasma membranes. The results suggest that these two sterols have opposite effects with respect to the ligand binding function of the receptor. Ergosterol was found to constrain the -opioid receptor in an inactive state in yeast plasma membranes and cannot replace cholesterol in activating it. These data differ from previous works dealing with the function of related G-protein-coupled receptors (GPCR) in ergosterol-enriched membranes. This suggests that structural requirements of GPCR with respect to their modulation by lipid components differ from one protein to another. As a consequence, we assume that the presence of appropriate lipids around transmembrane proteins determines their function. This highlights the functional significance of lateral heterogeneities of membrane components within biological membranes.To perform functional and structural studies of mammalian G-protein-coupled receptors (GPCR), 1 heterologous expression has long been a tool of choice (1). Unfortunately, although exceptions have been reported (2), these recombinant GPCR often fail to retain pharmacological properties similar to their native counterpart. Previous studies from our laboratory have focused on the expression of the human -opioid receptor in Saccharomyces cerevisiae (3). We found that the affinities of the antagonists were in the same range with yeast spheroplasts as in reference tissues. However those of agonists were shown to be lower. Assuming a lack of effective receptor coupling to endogenous G-protein ␣-subunits (i.e. GPA1 proteins (4, 5)), we were able to restore agonist binding upon addition of purified mammalian G-proteins (3). However, this approach requires high G-protein to receptor molar ratios and is not compatible with in vivo exploration of receptor functionality in heterologous systems. Finally, recent co-expressions of yeast/mammalian G-protein ␣-subunit chimeras with mammalian receptors have provided an alternative means to increase functional coupling (2, 6). According to this study and to restore human -opioid receptor activity in S. cerevisiae, we have co-expressed a G␣ i2 -GPA1 chimera protein with the receptor. Unfortunately, as further shown below, this approach failed to restore the ligand binding function of the -opioid receptor.Several investigations have suggested the importance of lipid membrane composition and organization in determining the activity of transmembrane proteins (7-11). Reports also emphasized the large diversity of lipid species found from one cell type to another (12, 13). Membrane composition and organization ...