Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes

Abstract

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. Methodology/Principal Findings: 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, t1/2 <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. Conclusion: 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. © 2009 Fagerholm et al.