Parathyroid hormone-induced alterations of protein content and phosphorylation in enriched apical membranes of opossum kidney cells

Abstract

Parathyroid hormone (PTH) reduces Na/Pi co-transport activity in opossum kidney (OK) cells in a process mediated by protein kinases A and C. Further, inactivation of Na/Pi transport involves irreversible inhibition, possibly via internalization, of the transport system. This study analyzed alterations of concentration and phosphorylation of membrane proteins of an apically enriched preparation induced by short (10 min) and long (3 h) term incubation with 10(-10) M PTH of monolayer cultures of the OK-cell line. To this end, an apically enriched membrane fraction was isolated from cells grown on Petri dishes and analyzed by two-dimensional gel electrophoresis. Long term exposure of the cells to PTH induced changes in apical protein concentration. Four proteins were found to be decreased and one protein was found to be increased in its concentration. Addition of 10(-10) M PTH to the cells led to transient phosphorylation of five proteins. In contrast to transient phosphorylation, phosphorylation of one protein increased over the time period of 3 h. Combined analysis of silver staining and autoradiography led to the detection of an acidic 35-kDa protein in which specific phosphorylation increased over a time period of hours. The results document for the first time alterations in apical membrane protein content and phosphorylation state mediated by PTH when added to an intact cellular system. It is concluded that the identified proteins represent possible candidates for being involved directly or indirectly in PTH alterations of membrane transport.