Purified Apical Membrane Endosomes from Rat Kidney Inner Medullary Collecting Duct Contain Endogenous Membrane-Bound Tyrosine Kinase (TK) and Phosphatase(TP) Activities • 1849

The epidermal growth factor receptor (EGFR) has been immunolocalized on the basolateral but not apical membranes of normal inner medullary collecting duct(IMCD) epithelial cells. In contrast, the epithelial cells of cysts arising from the IMCD in polycystic kidney disease display EGFR on both their apical and basolateral membranes. Activation of apical EGFR via TK signalling pathways is believed to play a role in cyst formation and progression. To determine if low levels of EGFR and/or endogenous TK or TP activities are present on the apical membrane of normal IMCD cells, we utilized a highly purified apical membrane endosome preparation from normal IMCD cells to perform immunoblotting studies described below. Immunoblots of apical membrane endosomes using anti-phosphotyrosine antibody reveal at least 5 basally-phosphorylated protein bands of approximately 90-93 kDa, 103-111 kDa, 120 kDa, 161 kDa and 171 kDa. The 161 kDa IMCD band comigrates with the EGFR band present in control A431 epithelial cells and is recognized by anti-EGFR antiserum suggesting that the 161 kDa IMCD band is an EGFR protein. Incubation of endosomes at 37°C in buffer containing 1 mM ATP and 1 mM MgCl₂ produced rapid dephosphorylation of all 5 phosphotyrosine bands. Phosphotyrosine dephosphorylation of all bands was inhibited by incubation at 4°C or 300 μM pervanadate (PV) but not 2 mM EDTA suggesting these TP(s) are not dependent on divalent cations. Coincubation of PV-treated endosomes with 100 μM A23 (a nonspecific TK inhibitor), significantly reduces the phosphotyrosine content of all 5 bands suggesting that both endogenous TK and TP are active under these conditions. Immunoblots of apical membrane endosomes also reveal they possess abundant caveolin-1 that has been demonstrated recently to inhibit EGFR TK activity (Couet, J. et al. J. Biol. Chem.272:30428, 1997). Triton X-100 extraction of endosomal proteins reveals that 120 kDa, 161 kDa and 171 kDa bands remain in the Triton X-100 insoluble fraction suggesting these proteins may be bound to caveolin-1. We conclude that purified apical membrane endosomes contain both endogenous TK and TP activities. Immunoblotting data suggest that a 161 kDa IMCD endosomal protein is EGFR that is a minor component of apical membrane endosomes where it is a substrate for TK and TP activity and may be bound to caveolin 1. These data serve as a basis for determination of the role(s) of apical membrane EGFR TK activity in normal vs polycystic kidney disease epithelia.