Depalmitoylation of rhodopsin with hydroxylamine

Intrinsic membrane proteins that to date have been investigated with respect to the function of palmitoylation are the beta-adrenergic receptor, rhodopsin, the alpha 2A-adrenergic receptor, and the influenza virus spike glycoprotein. As described above, the studies have led to differing conclusions with respect to the influence of palmitoylation on physiological activity. The basis of the differences remains unclear, but it may relate at least in part to the membrane environment of the protein during these studies, that is, the presence of a native membrane, the membrane composition of the expression cell line (in the case of mutant proteins), or the absence of membrane (in the case of detergent-purified proteins). For example, in the case of rhodopsin, the composition of the ROS disk membrane differs from that of the rod plasma membrane, and presumably also from the plasma membranes of cell lines in which mutant rhodopsins are expressed. Variation in membrane composition is known to have marked effects on the ability of rhodopsin to mediate the photic activation of PDE. Thus, although Karnik et al. clearly demonstrated the absence of an absolute requirement for palmitate in activating transducin, the influence of detergent on tertiary protein structure may have masked the full effect of the elimination of palmitate on the transducin-activating property of rhodopsin. Alternatively, the differing results obtained in the studies of rhodopsin could be a consequence of differences in amino acid sequences of the proteins studied. The precise functional role of the palmitate groups of rhodopsin remains an important question for further research. It was suggested by Ovchinnikov et al. that the hydrolysis of covalently bound palmitate might occur during the process of rhodopsin bleaching, but more recent data argue against this hypothesis. Experiments using synthetic peptides (representing cytoplasmic loop regions of rhodopsin) to identify the sites of interaction of R* and transducin provide support for an alternative possibility, namely, that palmitoylation and the resulting cytoplasmic loop play a role in the coupling of rhodopsin with transducin. The finding that the binding of transducin to R* occurs independently of the presence of palmitate argues against an essential requirement of palmitoylation on the binding step itself. However, available data indicate an enhancement, by depalmitoylation, of light-dependent GTPase activity in ROS preparations, although not in assays of unpalmitoylated, purified mutant rhodopsins (see above).(ABSTRACT TRUNCATED AT 400 WORDS)