Caveolae are subdomains of the plasma membrane which concentrate cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-linked proteins. It has recently been demonstrated that specific members of the Src family of protein tyrosine kinases require palmitoylation of NH2-terminal cysteine residues to localize in caveolae. Here we report that caveolin, an integral membrane protein which forms part of the coat of caveolae, also incorporates palmitate through linkage to cysteine residues. Caveolin contains only three cysteine residues which are all located on the COOH-terminal side of the hydrophobic transmembrane region. Immunofluorescent staining of cells transfected with caveolin indicated that, like the NH2 terminus, this COOH-terminal region is located on the cytoplasmic side of the plasma membrane. Studies of cysteine substitution mutants showed that all three cysteines are capable of incorporating palmitate and that the juxtamembrane Cys133 residue is the predominant site of palmitoylation. Simultaneous mutation of all three cysteine residues in caveolin resulted in the loss of ability to incorporate palmitate; however, this did not affect localization of the protein. Thus, palmitoylation of cysteine residues in nonmembrane spanning Src family protein tyrosine kinases has different consequences than in the transmembrane protein caveolin.
VIP21-caveolin is one of the components which form the cytoplasmic surface of caveolae. In vivo, this integral membrane protein is found in homo-oligomers with molecular masses of approximately 200, 400 and 600 kDa. These oligomers are also formed by the addition of eytosol to the in vitro synthesized and membrane inserted VIP21-caveolin. Here we show that long chain fatty aeyl eoenzyme A esters can completely substitute for cytosol in inducing 200 kDa and 400 kDa complexes, whereas 25-hydroxy-cbolesterol can produce the 200 kDa oligomer. In order to understand whether acylation of VIP21-eaveolin itself is a prerequisite for oligomerization, we studied a mutant protein lacking all three eysteines. When analyzed by velocity sucrose gradient centrifugation in the presence of the non-ionic detergent oetylglucoside, both palmitoylated and non-palmitoylated VIP21-caveolin formed oligomers that were indistinguishable. However, only the oligomers of the non-palmitoylated protein are disrupted when analyzed by SDS-PAGE without boiling. These data suggest that the protein domains of VIP21-eaveolin are the primary determinants of oligomerization, hut that palmitoylation of cysteine residues can increase the stability of the oligomers.
Neuroactive steroids modulate the function of ␥-aminobutyric acid, type A (GABA A ) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3␣,5-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA A receptors as evidenced by its ability to inhibit binding of [ 35 S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA A receptor subunits (␣ 1  2 ␥ 2 ). Certain endogenous pregnane steroids and their structural analogues are potent anesthetics in vertebrates (1, 2). These neuroactive steroids produce a rapid and reversible depression of the central nervous system indicating that their actions, unlike those of other steroid hormones, are not mediated by transcriptional regulation. In the 1980s it was demonstrated that neuroactive steroids could modulate the function of ␥-aminobutyric acid (GABA), 1 type A (GABA A ) receptors in the central nervous system (3-5). Low concentrations of the steroids potentiate the actions of GABA whereas higher concentrations directly open the GABA A receptor ion channel (6, 7). These observations led to the hypothesis that neuroactive steroids produce anesthesia by activating GABA A receptors and thus enhancing inhibitory synaptic transmission. The strong correlation between the ability of various neuroactive steroid analogues to modulate GABA A receptors and their ability to produce anesthesia strongly supports this hypothesis (8).The mechanism by which neuroactive steroids modulate GABA A receptor function remains unclear. In previous work we have tested the enantiomers of allopregnanolone (9) and pregnanolone (10) for their abilities to produce anesthesia and to modulate GABA A receptor function. These studies showed that both steroid anesthesia and steroid modulation of GABA A receptor function are highly enantioselective, particularly in the case of allopregnanolone. This indicates that neuroactive steroids most likely act via binding to specific recognition sites on the GABA A receptor protein complex, because the enantiomeric pairs have identical physical properties but mirror image shapes. Potentiation of GABA action by neuroactive steroids does not require any specific GABA A subunit (11) although the absence of the ␦ subunit does decrease the sensitivity of the receptor to neuroactive steroids (12). Radioligand binding studies and electrophysiological studies indicate that the putative neuroactive steroid binding sites are not identical to or overlapping with the identified binding sites for benzodiazepines (13), GABA (13) or picrotoxin (3) or to the putative binding site for barbiturates (13,14).A variety of anesthetic agents, including propofol, etomidate, benzodiazepines, and the halogenated alkanes and ethers, have also been shown to modulate GABA A receptor f...
We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-KB DNA-binding activity by T-cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1and interleukin 2-directed gene * Corresponding author.
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