Characterization of ANKRA, a Novel Ankyrin Repeat Protein that Interacts with the Cytoplasmic Domain of Megalin

Abstract

Abstract. 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.

Sequence-Specific Recognition of a PxLPxI/L Motif by an Ankyrin Repeat Tumbler Lock

“…The ANKRA2-binding peptide identified in HDAC4 is hydrophobic and contains several leucine and proline residues, which is reminiscent of a previously identified binding region for ANKRA2 on megalin. By means of yeast two-hybrid and glutathione S-transferase (GST)-mediated pull-down assays, Rader et al identified a leucine-and proline-rich, 19-residue fragment (HYRKTGSLLPTLPKLPSLS) from the cytoplasmic tail of megalin that bound to ANKRA2 (10). Therefore, we conclude that the binding sites for ANKRA2 on diverse proteins have related sequences.…”

“…Next, we examined whether the binding characteristics of the ANKRA2-HDAC4 complex were maintained in the interaction between ANKRA2 and megalin. The ANKRA2 binding site on megalin has been mapped to a leucine-and proline-rich 19-residue fragment (residues 4448 to 4466: HYRKTGSLLPTLPKLPSLS) (10). Because the ANKRA2-binding site on HDAC4 is formed by a PxLPxI sequence, we inspected the megalin sequence and found that not one but two overlapping PxLPxL motifs are embedded in this small region ( 4458 PTLPKL 4463 and 4461 PKLPSL 4466 , with consensus residues underlined; see Fig.…”

“…Ankyrin repeat family A protein 2 (ANKRA2) is an ankyrin repeat domain-containing protein and was first identified as a binding partner for the cytoplasmic domain of megalin, which is also known as low-density lipoprotein receptor-related protein 2 (LRP2) (10). Megalin is a multiligandbinding receptor found in the plasma membrane of many absorptive epithelial cells, and it regulates the endocytosis of various extracellular signaling proteins (11).…”

“…Megalin is a multiligandbinding receptor found in the plasma membrane of many absorptive epithelial cells, and it regulates the endocytosis of various extracellular signaling proteins (11). ANKRA2 is thought to play a regulatory role in the function of megalin as a clearance receptor in the kidney and in L2 cells (10), and through yeast two-hybrid screening, ANKRA2 was identified as a binding partner of histone deacetylase 4 (HDAC4) and HDAC5 (12,13). HDAC4 and HDAC5 are members of the class IIa subgroup of the HDAC superfamily and mainly function as co-repressors for DNA binding transcription factors, such as myocyte enhancer factor 2 (MEF2) family members, to repress gene expression in diverse developmental programs (14,15).…”