FC Binding by Proteins Including Caveolin

In contrast to reviews of protein-protein and protein-DNA binding, very few studies have been made of the structural aspects of FC binding to proteins. The sum of evidence available suggests that FC lies within a hydrophobic cleft or tunnel, the sides of which are composed of several different hydrophobic sequences organized by the tertiary structure of the native protein. A positively charged amino acid (K or R) may contribute to a "charge clamp" stabilizing the 3P-OH group. Whilst the volume of FC has been estimated at 741 A3 [91], its binding cavity is usually about twice as large. There is evidence in some instances that FC itself induces significant changes in the shape and size of this cavity. In the case of many of the FC-binding proteins for which a tertiary structure has been established, one of the hydrophobic sequences contributing to the FC-binding cavity conforms to the "cholesterol recognition" consensus motif (-V/Lx1-4 Yx1-4 K/R-) [86] (Table 5.1).

The caveolin central domain (cav82-101) also contains a cholesterol recognition sequence (CRAC-1) (Fig. 5.3B) that binds FC [96]. The isolated peptide also binds FC from FC/phosphatidylcholine vesicles to an extent that exceeds FC solubility in the lipid bilayer. This result, based on tryptophan fluorescence and NOESY data, indicates the ability of the peptide to induce macromolecular order in FC, possibly by stabilizing FC-FC links [97]. The sequence required for FC binding (-V94TKYWFYR-) abuts the residues in cav(82-101) needed for interaction with signal proteins Gi2a [80] and eNOS [76]. A key question considered later is whether the binding of protein and FC to the same domain is complementary or competitive.

Although residues 94-101 within the caveolin(83-102) peptide promote binding with synthetic FC-containing liposomes, it seems likely that additional hydrophobic residues contribute to membrane binding by caveolin in vivo. Phosphorylation at S80 has a substantial effect on FC binding [50], suggesting a role for neighboring hydrophobic residues within the sequence -S80FDGLW-. In contrast, phosphorylation at Y14 completely inhibited FC binding, which may indicate that the short hydrophobic sequence -L13YTV- may also contribute. Though the data remain limited, it seems likely that the structural basis of FC binding in caveolin resembles that in other proteins.

Do other proteins present in caveolae bind FC? A number of caveolin-associated proteins crosslink to the photoactivable FC analogue FCBP [13], and many caveolin-associated proteins including eNOS and PDGFR include one or more "cho-

Table 5.2 Effects of FC depletion (Y) and cav (82-101) on activities of caveolar proteins.

Activity

Effect of FC Y

Addition of cav (82-101)

Reference(s)

Neutral ceramidase

X

109

Adenyl cyclase

Y

Y

90

Ca2+-act K+ channel

t

64

TGFbeta-1 receptor

t

110

Ca2+-dependent PLA2

Y

Y

111, 112

Ca2+-ATPase

Y

113

ERK1/2

Y

114

p-glycoprotein

Y

115

Agonist stim cAMP accum

t

116

Insulin receptor

Y

117

eNOS

Y

Y

80, 118

PKCa translocation

Y

Y

119, 120

FC was depleted from cultured cells with beta-methyl cyclodextrin. Cav (82-101) was determined either in vitro in cultured cell homogenates, or in vivo as a complex with a cell-permeable antennopedia peptide.

FC was depleted from cultured cells with beta-methyl cyclodextrin. Cav (82-101) was determined either in vitro in cultured cell homogenates, or in vivo as a complex with a cell-permeable antennopedia peptide.

lesterol recognition" motifs within the primary sequence, though at present experimental data showing if these sequences are involved in FC binding are lacking.

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