Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides.
How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol-anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle-induced clusters of both Thy-1 and CD73, a 5' exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.
Chen
Yun
Y
Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Thelin
William R
WR
Yang
Bing
B
Milgram
Sharon L
SL
Jacobson
Ken
K
D015720:5'-Nucleotidase / Q000378:metabolism; D000818:Animals; D051242:Caveolin 1 / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002784:Cholesterol / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006046:Gold / Q000032:analysis; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D053758:Nanoparticles / Q000032:analysis; D010716:Phosphatidylinositols / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D018800:Thy-1 Antigens / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D051242:Caveolin 1; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D018800:Thy-1 Antigens; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold; D002784:Cholesterol; D019061:src-Family Kinases; D015720:5'-Nucleotidase