Podocalyxin activates RhoA and induces actin reorganization through NHERF1 and Ezrin in MDCK cells

Abstract

Podocalyxin (PC) is the major sialoglycoprotein expressed on the apical membrane of the podocyte. Previously it was shown that PC is connected to actin through the PC/NHERF2/ezrin complex, and this connection is disrupted in the nephrotic syndrome. For assessing whether expression of PC affects the organization of the actin cytoskeleton, MDCK cell lines stably expressing either full-length PC or a PC mutant lacking the NHERF binding site was established. It was found that full-length PC but not the PC mutant is connected to actin, induces redistribution of actin toward the apical membrane, and leads to increased RhoA activity. By immunofluorescence redistribution of RhoA and RhoGDI was observed in the presence of both full-length PC and the PC mutant. With the use of pulldown assays, PC and ezrin were found to interact directly and the ezrin binding site was mapped to the juxtamembrane region of PC's cytoplasmic tail. It is concluded that PC binds to ezrin both directly and indirectly. PC activates RhoA through NHERF and ezrin, leading to redistribution of actin filaments. These results suggest that in podocytes, PC may also regulate foot process architecture through RhoA.