AN INTACT AND FUNCTIONAL SOLUBLE FORM OF THE INSULIN-RECEPTOR IS SECRETED BY CULTURED-CELLS

We have recently reported that the insulin receptor (IR), a tetrameric transmembrane protein located on the surface of target cells, is pres ent as a soluble form in human plasma. In the present study we investi gated whether human cells in culture release an intact and functional form of the IR. We found that IRs are secreted into the incubation med ium by four cell lines (IM-9 human lymphoblasts, MCF-7 human breast ca ncer cells, HepG2 human hepatoma cells, and 3T3 mouse fibroblasts tran sfected with human IRs). IR secretion was further characterized in IM- 9 cells. IR release was time, temperature, and energy dependent, and e nhanced by incubation with insulin. The dilution slope of secreted IRs in a specific IR RIA was parallel to that produced by highly purified human placenta IRs. Ligand binding studies revealed that secreted rec eptors bound insulin with high affinity, and the Scatchard analysis re vealed two orders of binding sites (the high affinity site had a disso ciation constant of 0.32 +/- 0.08 nm). Analysis of secreted receptors by sodium dodecyl sulfatepolyacrylamide gel electrophoresis demonstrat ed a molecular size of 135 kilodaltons for the a-subunit and 95 kiloda ltons for the beta-subunit. Other experiments indicated that the beta- subunit tyrosine kinase activity of the secreted receptor was stimulat ed by insulin. These studies indicate, therefore, that a soluble, inta ct, and functional IR is secreted by cultured cells, and that this sol uble protein could be involved in certain insulin-mediated functions, such as receptor down-regulation.