RESUMO
Ankyrin-repeat family A protein (ANKRA) is a novel protein that interacts directly and specifically with the cytoplasmic tail of megalin in the yeast two-hybrid system and glutathione-S-transferase pull-down assays. ANKRA has three ankyrin repeats and shows 61% overall homology to regulatory factor X, ankyrin repeat-containing protein. Mapping studies show that the three ankyrin repeats and C-terminus of ANKRA are required for binding to a unique juxtamembrane, 19-amino acid sequence on the megalin tail. Point mutational analysis reveals that a proline-rich motif (PXXPXXP) within this region is the site of ANKRA binding. ANKRA interacts with megalin but not with low-density lipoprotein receptor related protein, in keeping with the fact that the sequence of the megalin tail is unique. By cell fractionation, ANKRA is found both in the cytosol and associated with membranes enriched in megalin in L2 cells and proximal tubule cells. By immunofluorescence, ANKRA is concentrated near megalin along the plasma membrane of L2 cells and in the kidney cortex is expressed in glomerular and proximal tubule epithelia which also express megalin. These observations suggest that ANKRA may play a unique role in megalin's function as a clearance receptor in the kidney and L2 cells. In addition, ANKRA may have other partners because northern blot analysis reveals that ANKRA is more broadly expressed than megalin, and by immunofluorescence ANKRA is also expressed in connecting tubule cells and principal cells of collecting ducts.
