FC in Caveolae Effects of Depletion and Loading

It is characteristic of proteins in caveolae that their activities are very sensitive to membrane levels of FC. In some cases these activities are increased, in other cases inhibited, when FC is depleted (Table 5.2). Addition of the cav(82-101) peptide -either in vitro or in vivo - spliced to an antennopedia (AP) "Trojan peptide" [78] also affects the activity of caveola-associated signaling proteins. In those examples where comparative data are available, the addition of peptide (at 10-100 mM) in each case mimics the effect of FC depletion. Is this coincidence, or informative about the role of FC in this system?

One way to look at the role of FC in caveolar signaling is to modify the level of FC, and to determine the effects of this on the activity of caveola-associated proteins. Much of the research on the influence of FC on caveolae has been carried out with cyclodextrins, cyclic oligosaccharides containing six to eight a-1,4-linked-glycopyranoside units enclosing a central hydrophobic tunnel [98]. These bind

Fig. 5.4 Alternative mechanisms for the effects of caveolin(82-101) peptide on functional multiprotein complexes in caveolae. Mechanisms shown: A = displacement of caveolin from signaling kinase (PDGFR) by anteonnopedia (AP)-linked caveolin(82-101) peptide. B = displacement of PDGFR from its association with caveolin by AP peptide binding to caveolin. C = the same AP-peptide acting as a sink for caveolar FC.

Fig. 5.4 Alternative mechanisms for the effects of caveolin(82-101) peptide on functional multiprotein complexes in caveolae. Mechanisms shown: A = displacement of caveolin from signaling kinase (PDGFR) by anteonnopedia (AP)-linked caveolin(82-101) peptide. B = displacement of PDGFR from its association with caveolin by AP peptide binding to caveolin. C = the same AP-peptide acting as a sink for caveolar FC.

small hydrophobic molecules in a reaction depending on the replacement of core water molecules by the organic ligand. b-Methyl and b-hydroxypropyl cyclodextrins have an internal cavity of ~0.8 nm. They interact with FC, but not with other common membrane lipids. Consequently, FC-free cyclodextrins are effective and selective receptor-independent acceptors of cell FC by diffusion. Conversely, preformed cyclodextrin-FC complexes, or low-density lipoprotein (LDL), can donate FC to increase levels of FC-rich microdomains, including caveolae. Decreased FC mediated by cyclodextrin led to the dissociation of signaling proteins from cav-eolae, disassembly of caveolin multimers [10] and eventually, to a reduction in the rate of caveolin synthesis by transcriptional down-regulation [99]. In human primary fibroblasts and smooth muscle cells the chronic effects of FC loading included an increase in cell-surface caveolae by transfer of caveolin from intracel-lular pools [49], increased endocytosis of FC-rich vacuoles [100] and, over time, the induction of caveolin synthesis at the transcriptional level by a FC-sensitive promoter site [101,102]. In human primary endothelial cells, an increase in caveolin at the cell surface in response to LDL was drawn mainly from intracellular pools [103]. These data suggest that the presence of FC is closely linked to both the structure and function of caveolae.

Three different mechanisms could give rise to the displacement by caveolin peptide(82-101) of signaling proteins from their complex with caveolin (Fig. 5.4):

1. Caveolin peptide(82-101) competes with the target kinase, binding caveolin and generates free kinase.

2. Caveolin peptide(82-101) competes with caveolin to bind the target kinase.

3. Caveolin peptide(82-101) binds FC competitively, displacing it from caveolin, secondarily dissociating the caveolin-kinase complex.

Since peptides cav(1-101) and cav(1-79) polymerize similarly in vitro [45], FC-binding to cav (82-101) [96] is unlikely to play a direct role in caveolin self-assembly. These data argue against the first hypothesis. Since the cav(82-101) peptide mimics caveolin and, like the full-length protein, binds FC, it is difficult to envisage a mechanism by which cav(82-101) would reverse the effect of native caveolin. Other evidence supports the hypothesis that cav(82-101) may mimic cyclo-dextrin by binding FC competitively. The relatively high concentrations of this freely internalized peptide required for maximal effect (50-100 mM) would be adequate to bind significant amounts of plasma membrane FC. F92 of intact caveolin is necessary for the effect of peptide on eNOS [80], and the same residue is required for FC binding [96]. We suggest that the scaffold peptide cav(82-101) may act at least in part to competitively sequester caveolar FC.

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