Sterol efflux to apolipoprotein A-I originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activity.
The kinetics of sterol efflux from human aortic smooth muscle cells equilibrated with a [(3)H]benzophenone-modified photoactivable free cholesterol analogue ((3)H-FCBP) did not differ significantly from those labeled with free cholesterol ((3)H-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 (3)H-FC and (3)H-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 approximately 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 (3)H-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.
Fielding
P E
PE
Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California 94143, USA. pefield@itsa.ucsf.edu
Russel
J S
JS
Spencer
T A
TA
Hakamata
H
H
Nagao
K
K
Fielding
C J
CJ
D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*; D013696:Temperature; D014357:Trypsin
D016632:Apolipoprotein A-I; D051242:Caveolin 1; D022461:Caveolins; D010982:Platelet-Derived Growth Factor; D013261:Sterols; D002784:Cholesterol; D011505:Protein-Tyrosine Kinases; D014357:Trypsin