PODOCALYXIN (PODXL) DIFFERENT DOMAINS HAVE CELLULAR AND EXTRACELLULAR FUNCTIONS

FERNÁNDEZ, DARÍO; AYUSO, MATILDE

Granada

Congreso; XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular; 2014

Sociedad Española de Bioquímica y Biología Molecular

CHO cells were stably transfected with cDNA encoding a fusion protein of PODXL and green fluorescence protein (PODXL-GFP). Soon after seeding, PODXL-GFP was detected in vivo, by widefield microscopy, in the Golgi and later on as microvesicles, randomly distributed all over the cytoplasm and at the plasma membrane. After 10 h of incubation, the PODXL microvesicles were preferentially located at the leading edges of the cell, progressing along the filopodia, to finally be released into the extracellular medium. CHO-PODXL-GFP cells showed the presence of two distinct anti-PODXL immunoreactive bands of ~200 and ~160 KDa, respectively. The first one reacted with anti-GFP whereas the smaller one did not, suggesting the absence of the carboxyterminal domain of PODXL. Those two bands were also detected in the insoluble fraction of extracellular medium and were identified as PODXL-GFP or PODXL-Δ by mass spectrometry analysis. An additional anti-PODXL reactive form, of ~ 70 KDa was also detected in the soluble extracellular fraction. Either intact or truncated forms of immunoreactive PODXL increased in the insoluble or soluble fractions of extracellular medium upon incubation of CHO-PODXL-GFP cells with PMA, effect that was prevented by either PKC inhibitors, or matrix metalloproteinase inhibitors. It is concluded that PODXL is released from the cell mainly as a cargo of microvesicles and also as a soluble cleaved fragment of the PODXL ectodomain. Thus, in studying the cellular actions of PODXL, the combined action of membrane-bound and free (truncated) forms of this protein should be considered.