FUNCTION AND DIFFERENTIAL REGULATION OF THE ALPHA-6 INTEGRIN ISOFORMSDURING PARIETAL ENDODERM DIFFERENTIATION

F9 embryonal carcinoma cells treated with retinoic acid differentiate in monolayer into parietal endoderm (PE) or in suspension into embryoi d bodies with an outer layer of visceral endoderm (VE) surrounding a c ore of largely undifferentiated cells. Previous reports have shown tha t cell-extracellular matrix interactions mediated by the beta 1 integr ins play a critical role in the differentiation and migration of PE. I n the present study we investigated the pattern of expression and func tion of the integrin alpha 6 beta 1 during the differentiation of F9 c ells into VE and PE. F9 cells express integrin subunits alpha 3, alpha 5, alpha 6, and beta 1. Cell adhesion and migration assays demonstrat e that alpha 6 beta 1 is the major laminin receptor in undifferentiate d F9 cells as well as F9-derived PE cells. However, the amount of alph a 6 protein decreases significantly upon F9 cell differentiation into either VE or PE, as revealed by immunofluorescent staining and immunop recipitation analysis. In contrast, the amount of steady-state alpha 6 message stays constant before and after F9 cell differentiation, sugg esting that the downregulation of alpha 6 beta 1 occurs post-transcrip tionally. In view of previous reports of two alpha 6 isoforms generate d by alternative RNA processing, we carried out reverse transcription- PCR analysis and show that, while alpha 6B is the major mRNA isoform b efore and after F9 cell differentiation, alpha 6A mRNA is weakly expre ssed in undifferentiated F9 cells and is substantially increased follo wing F9 differentiation into PE. Immunoprecipitations using the isofor m-specific antibodies show an increase in alpha 6A and a dramatic decr ease in alpha 6B protein following PE differentiation. Pulse-chase exp eriments indicate that, whereas the stability of alpha 6B protein is u naltered, synthesis of alpha 6B protein is decreased at least threefol d following PE differentiation. Further experiments demonstrate that a lpha 6A localizes to focal contacts in PE cells. The switch from alpha 6B to alpha 6A and the localization of alpha 6A at focal contacts cor relate with the acquisition of PE cell motility, which suggests distin ct functions for the two alpha 6 isoforms. (C) 1995 Academic Press, In c.