ADAM10 contributes to transcriptional regulation of megalin.

Megalin is subjected to regulated intramembrane proteolysis (RIP) that may define a novel signaling pathway in proximal tubule (Zou et al., JBC 279:34302, 2004). RIP includes metalloprotease‐mediated ectodomain shedding and release of megalin's cytosolic domain by γ‐secretase. To identify brush border metalloproteases that may participate in this pathway we examined the renal distribution of ADAM10, known to mediate ectodomain shedding of other receptors subjected to RIP. Indirect immunofluorescence microscopy and immunoblotting of renal microsomal fractions showed ADAM 10 localized to the brush border of the proximal tubule. To study the role of ADAM10 activity in the proximal tubule we stably over‐expressed ADAM10, inactive ADAM10 (ADAM10dn) or empty vector in OKP cells. ADAM10dn was rendered inactive by a point mutation (E384A) in the zinc‐binding pocket. Both ADAM10 and ADAM10dn transfected cells expressed comparable levels of transfected protein as determined by immunoblotting. All cell lines appeared normal and had the same total protein profile as seen by SDS/PAGE and coomassie blue staining. However, expression of specific apical membrane proteins (megalin, NHE3, Dab2) were 3–5 fold lower in ADAM10dn cells compared to ADAM10 transfected or control cells. We also found a dramatic reduction in megalin mRNA in cells expressing ADAM10dn compared to the other cells. In conclusion, these data show that ADAM10 activity regulates megalin gene expression in proximal tubule. We postulate that the metalloprotease activity mediated by ADAM10 is part of a signaling pathway in proximal tubule involved in transcriptional regulation of brush border gene products.