Sterol efflux to apolipoprotein AI originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activity

PE Fielding, JS Russel, TA Spencer, H Hakamata… - Biochemistry, 2002 - ACS Publications
PE Fielding, JS Russel, TA Spencer, H Hakamata, K Nagao, CJ Fielding
Biochemistry, 2002ACS Publications
The kinetics of sterol efflux from human aortic smooth muscle cells equilibrated with a [3H]
benzophenone-modified photoactivable free cholesterol analogue (3H-FCBP) did not differ
significantly from those labeled with free cholesterol (3H-FC). Trypsin digestion of caveolin
cross-linked by photoactivation of FCBP led to association of radiolabel in a single low
molecular weight fraction, indicating relative structural homogeneity of caveolin-bound
sterol. These findings were used to investigate the organization of sterols in caveolae and …
The kinetics of sterol efflux from human aortic smooth muscle cells equilibrated with a [3H]benzophenone-modified photoactivable free cholesterol analogue (3H-FCBP) did not differ significantly from those labeled with free cholesterol (3H-FC). Trypsin digestion of caveolin cross-linked by photoactivation of FCBP led to association of radiolabel in a single low molecular weight fraction, indicating relative structural homogeneity of caveolin-bound sterol. These findings were used to investigate the organization of sterols in caveolae and the ability of these domains to transfer sterols to apolipoprotein A-I (apo A-I), the major protein of human plasma high-density lipoproteins (HDL). During long-term (4−5 h) incubation with apo A-I, caveolin-associated 3H-FC and 3H-FCBP decreased, in parallel with an increase in apo A-I-associated sterol. Assay of caveolin-associated labeled sterols indicated that caveolae were a major source of sterol lost from the cells during HDL formation. Short-term changes of sterol distribution in caveolae were assayed using platelet-derived growth factor (PDGF). PDGF was without effect on FC efflux in the absence of apo A-I, but when apo A-I was present, PDGF increased FC efflux ∼3-fold beyond the efflux rate catalyzed by apo A-I alone. At the same time, caveolin-associated FC decreased, and PDGF-dependent protein kinase activity was stimulated. Parallel results were obtained with 3H-FCBP-equilibrated cells, in which apo A-I potentiated a PDGF-mediated reduction of radiolabel cross-linked to caveolin following photoactivation. These results suggest that sterols within caveolae are mobile and selectively transferred to apo A-I. They also suggest a novel role for sterol efflux in amplifying PDGF-mediated signal transduction.
ACS Publications