Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes.
BACKGROUND
The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.


RESULTS
We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t(1/2)<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.


CONCLUSIONS
It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.
Fagerholm
Siri
S
Division of Cell Biology, Department of Clinical and Experimental Medicine and Diabetes Research Centre, University of Linköping, Linköping, Sweden.
Ortegren
Unn
U
Karlsson
Margareta
M
Ruishalme
Iida
I
Strålfors
Peter
P
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002746:Chlorpromazine / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D007328:Insulin; D011990:Receptors, Transferrin; D014638:Vanadates; D011972:Receptor, Insulin; D002746:Chlorpromazine