Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol.
Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mbeta-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-alpha activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCalpha, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.
Sharma
Deepak K
DK
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Brown
Jennifer C
JC
Choudhury
Amit
A
Peterson
Timothy E
TE
Holicky
Eileen
E
Marks
David L
DL
Simari
Robert
R
Parton
Robert G
RG
Pagano
Richard E
RE
D016227:Benzoquinones; D021941:Caveolae / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology* / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006028:Glycosphingolipids / Q000494:pharmacology* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D047029:Lactams, Macrocyclic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051571:Protein Kinase C-alpha; D011743:Pyrimidines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C412373:AG 1879; D016227:Benzoquinones; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D006028:Glycosphingolipids; D047029:Lactams, Macrocyclic; D011743:Pyrimidines; D011809:Quinones; D049452:Green Fluorescent Proteins; D017828:Rifabutin; C020754:herbimycin; D002784:Cholesterol; D019061:src-Family Kinases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha