ARTICLES J Am Soc Nephrol 11: 2167–2178, 2000 Characterization of ANKRA, a Novel Ankyrin Repeat Protein that Interacts with the Cytoplasmic Domain of Megalin KATHERINE RADER,* ROBERT A. ORLANDO,† XIAOJING LOU,* AND MARILYN GIST FARQUHAR*† Departments of *Cellular and Molecular Medicine and †Pathology, University of California San Diego, La Jolla, California. Abstract. Ankyrin-repeat family A protein (ANKRA) is a unique. By cell fractionation, ANKRA is found both in the novel protein that interacts directly and specifically with the cytosol and associated with membranes enriched in megalin in cytoplasmic tail of megalin in the yeast two-hybrid system and L2 cells and proximal tubule cells. By immunofluorescence, glutathione-S-transferase pull-down assays. ANKRA has three ANKRA is concentrated near megalin along the plasma mem- ankyrin repeats and shows 61% overall homology to regulatory brane of L2 cells and in the kidney cortex is expressed in factor X, ankyrin repeat-containing protein. Mapping studies glomerular and proximal tubule epithelia which also express show that the three ankyrin repeats and C-terminus of ANKRA megalin. These observations suggest that ANKRA may play a are required for binding to a unique juxtamembrane, 19-amino unique role in megalin’s function as a clearance receptor in the acid sequence on the megalin tail. Point mutational analysis kidney and L2 cells. In addition, ANKRA may have other reveals that a proline-rich motif (PXXPXXP) within this region partners because northern blot analysis reveals that ANKRA is is the site of ANKRA binding. ANKRA interacts with megalin more broadly expressed than megalin, and by immunofluores- but not with low-density lipoprotein receptor related protein, in cence ANKRA is also expressed in connecting tubule cells and keeping with the fact that the sequence of the megalin tail is principal cells of collecting ducts. Megalin (approximately 600 kD), a member of the low-density families, plasminogen, lactoferrin, thyroglobulin, Ca2ϩ, and lipoprotein receptor (LDLR) superfamily, is found in clathrin- receptor-associated protein (RAP), a chaperone for members of coated vesicles in glomerular and proximal tubule epithelial the LDLR gene family (1,3). cells of the kidney (1). Recent studies show that megalin is the Although considerable work has been done on ligand bind- main endocytic receptor in the proximal tubule and specifically ing to the extracellular domain of megalin, the role of its binds and internalizes a number of proteins that are filtered by cytoplasmic domain in regulating megalin’s endocytic or exo- the glomerulus (2,3). These proteins include insulin (2); apo- cytic trafficking pathways (1,6) or putative Ca2ϩ-sensing func- lipoprotein H/I complexes; carriers for vitamins D3,B12, and A tion (7) is completely unknown. Megalin has a unique cyto- (retinol); and parathyroid hormone, among others (3,4). Mega- plasmic tail that shows little sequence similarity to other LDLR lin knockout mice exhibit proximal tubule reabsorption defi- family members except for conserved NPXY motifs (8). The ciency and a significant reduction in the number and size of fact that the cytoplasmic tail of receptors regulates their traf- organelles (clathrin-coated pits, endosomes, dense apical tu- ficking and signaling suggests that megalin has trafficking bules, and lysosomes) associated with endocytosis in the prox- itineraries or intracellular signaling pathways distinct from imal tubule (3,4). Thus, megalin expression is directly corre- those of other LDLR family members. The presence of lated with endocytic activity in the proximal tubule. In other dileucine, YXXØ, PXXP, and (S/T)XV motifs as well as systems, megalin has been shown to bind a number of other NPXY motifs, provides clues to the function of the megalin tail ligands from the lipoprotein and protease/protease inhibitor (7,8). Tyrosine-based motifs (NPXY, YXXØ) and dileucine motifs serve to direct receptors to clathrin-coated pits for rapid internalization or sorting within the trans-Golgi network by binding selectively to adaptor proteins AP-1, AP-2, or AP-3 Received February 22, 2000. Accepted May 30, 2000. Data deposition: The sequences reported in this article have been deposited in (9–11). This is in keeping with the fact that megalin, like other the GenBank database (Accession nos. hANKRA, AF314032; mANKRA, members of the LDL family, is internalized via clathrin-coated AF314031). pits (1,6,12). PXXP motifs bind to SH3 domains, (S/T)XV to Correspondence to Dr. Marilyn Gist Farquhar, Department of Cellular and PDZ domains, NPXY to PTB domains, and YXXØ to SH2 Molecular Medicine, University of California San Diego, 9500 Gilman Drive, domains (13). These interactions have been shown to be im- La Jolla, CA 92093-0651. Phone: 858-534-7711; Fax: 858-534-8549; E-mail: [email protected] portant for subcellular localization (14,15), endocytic traffick- 1046-6673/1112-2167 ing (16–19), and signaling (13–15,20) of some transmembrane Journal of the American Society of Nephrology proteins. However, nothing is known concerning the functions Copyright © 2000 by the American Society of Nephrology or molecular interactions of these motifs in the case of megalin. 2168 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 2167–2178, 2000 To gain insight into the interactions of the megalin tail and in 10% fetal calf serum (Life Technologies, BRL, Grand Island, NY), thereby into megalin’s functions, we used the two-hybrid sys- MEM-alpha medium (Irvine Scientific, Santa Ana, CA). tem to identify proteins that bind to the cytoplasmic domain of megalin and therefore might be involved in directing megalin Library Screening and DNA Sequencing traffic or signaling. In this article, we report the initial charac- The cDNA encoding the cytoplasmic domain of rat megalin was terization and localization of a novel protein, ANKRA, which obtained by PCR from clone 217 (8) and cloned into the yeast specifically binds to the cytoplasmic domain of megalin. two-hybrid pGBT9 “bait” vector (Clontech). To isolate proteins that interact with the megalin tail, we screened a mouse kidney oligo-dT and random-primed MATCHMAKER cDNA library (Clontech) using Materials and Methods the yeast two-hybrid system as described previously (27,28). Of 5 cDNA and Antibodies million transformants, three scored positive (blue) during lacZ selec- Two incomplete expressed sequence tags (ESTs) of human tion and were found to be identical by restriction digest grouping and ANKRA were obtained from the I.M.A.G.E. Consortium (Washing- sequencing the 5' ends. The insert contained the C-terminal half of ton University-Merck EST project, L. Hillier, unpublished data) and ANKRA (bp 745 to 1774) encoding aa138 to 312, the 3'UTR, and the sequenced by automated DNA sequencing. A complete human cDNA poly-A tail. To obtain the full-length cDNA, a mouse pituitary random sequence (1507 bp long) including the poly A tail was assembled from primed ␭gt-11 cDNA library (custom made by Stratagene and ob- overlapping human ESTs (GenBank accession numbers aa418089, tained from G. M. Rosenfeld, University of California San Diego) was aa442702, N6431, and AW079850). A plasmid containing the entire screened with a cDNA probe encoding aa138 to 312 of ANKRA sequence of human low-density lipoprotein receptor related protein according to the library manufacturer’s instructions. Three clones (LRP) was obtained from Joachim Herz (University of Texas were obtained: clone 11 encoded bp 1 to 1646 of ANKRA including Southwestern). the 5'UTR and 3'UTR, and clones 2 (full-length) and 3 (partial) were Rabbit megalin antiserum (459) was raised against a peptide from alternatively spliced in the C-terminus and 3'UTR, respectively. the cytoplasmic tail of megalin as described (21). Mouse monoclonal Clones were sequenced by either the dideoxynucleotide chain-termi- (mAb) anti-megalin IgG 20B was prepared as described previously nation method with Sequenase (United States Biochemical, Cleve- ␤ (22). Polyclonal anti- 1 integrin (130) was a generous gift from land, OH) or automated sequencing (DNA Sequencing Facility, Richard O. Hynes (MIT). The following antibodies were purchased: Scripps Research Institute, San Diego, CA). anti-HA mAb (BAbCO), goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (HRP; Pierce, Rockford, IL), Amino Acid Sequence Analysis and Northern and cross-absorbed goat anti-rabbit Alexa 488 or anti-mouse Alexa Blot Analysis 594 F(ab')2 (Molecular Probes, Eugene, OR). Antisera were raised in rabbits against purified histidine (His)- On-line BLAST searches were performed via the National Center tagged full-length ANKRA and were affinity purified by coupling for Biotechnology Information at the National Institutes of Health purified His-tagged ANKRA (1-181) to AffiGel 15 beads (BioRad, (Bethesda, MD) (27). Scans for biologically significant sites, patterns, Hercules, CA) according to the manufacturer’s instructions. Bound and localization motifs on ANKRA were performed on-line using IgG was eluted with 0.1 M glycine (pH 2.5). By immunoblotting, 0.6 PROSITE (Swiss-Institute of Bioinformatics) and PSORT (Institute ␮g/ml affinity-purified anti-ANKRA (1-181) IgG detected 10 ng for Molecular and Cellular Biology, Osaka University, Osaka, Japan). purified His-tagged ANKRA. Sequence alignment was done using Clustal W and MacBoxShade ([email protected]) software programs. A Northern blot from eight murine tissues containing 2 ␮g poly Expression and Purification of Fusion Proteins Aϩ RNA per lane was purchased from Clontech and hybridized with Sequences of mouse ANKRA were subcloned by PCR into 32P-labeled probes prepared as described (28). pET28a(ϩ) vector (Novagen, Madison, WI). His-tagged fusion pro- teins were expressed in BL21(DE3) bacteria (Stratagene, La Jolla, GST-Precipitation Assays from Cell Lysates CA). Because the fusion proteins were insoluble in 1% Tween-20, Mouse ANKRA was subcloned by PCR behind an HA-epitope in they were solubilized from inclusion bodies with 8 M urea (in 20 mM the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, Tris [pH 7.8], 0.5 M NaCl, and 5 mM Imidazole), bound to Ni-NTA CA).