E-cadherin polarity is determined by a multifunction motif mediating lateral membrane retention through ankyrin-G and apical-lateral transcytosis through clathrin.

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.

Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protection of skeletal muscle from exercise injury.

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with ß2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on ß2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required ß2 spectrin. Together, these results are consistent with a functional hierarchy beginning with ß2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.

An ankyrin-based mechanism for functional organization of dystrophin and dystroglycan.

beta-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an ankyrin-based mechanism for sarcolemmal localization of dystrophin and beta-DG. Dystrophin binds ankyrin-B and ankyrin-G, while beta-DG binds ankyrin-G. Dystrophin and beta-DG require ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and beta-DG remain intracellular in ankyrin-B-depleted muscle, where beta-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires ankyrin-B for localization of dystrophin/utrophin and beta-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4.

Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos.

E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.

Isoform specificity of ankyrin-B: a site in the divergent C-terminal domain is required for intramolecular association.

Ankyrins contain significant amino acid identity and are co-expressed in many cell types yet maintain unique functions in vivo. Recent studies have identified the highly divergent C-terminal domain in ankyrin-B as the key domain for driving ankyrin-B-specific functions in cardiomyocytes. Here we identify an intramolecular interaction between the C-terminal domain and the membrane-binding domain of ankyrin-B using pure proteins in solution and the yeast two-hybrid assay. Through extensive deletion and alanine-scanning mutagenesis we have mapped key residues for interaction in both domains. Amino acids (1597)EED(1599) located in the ankyrin-B C-terminal domain and amino acids Arg(37)/Arg(40) located in ANK repeat 1 are necessary for inter-domain interactions in yeast two-hybrid assays. Furthermore, conversion of amino acids EED(1597) to AAA(1597) leads to a loss of function in the localization of inositol 1,4,5-trisphosphate receptors in ankyrin-B mutant cardiomyocytes. Physical properties of the ankyrin-B C-terminal domain determined by circular dichroism spectroscopy and hydrodynamic parameters reveal it is unstructured and highly extended in solution. Similar structural studies performed on full-length 220-kDa ankyrin-B harboring alanine substitutions, (1597)AAA(1599), reveal a more extended conformation compared with wild-type ankyrin-B. Taken together these results suggest a model of an extended and unstructured C-terminal domain folding back to bind and potentially regulate the membrane-binding domain of ankyrin-B.

Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain.

We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B(+/-) cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

Isoform specificity among ankyrins. An amphipathic alpha-helix in the divergent regulatory domain of ankyrin-b interacts with the molecular co-chaperone Hdj1/Hsp40.

Ankyrins-R, -B, and -G are a family of membrane-associated adaptors required for localization of structurally diverse proteins to specialized membrane domains, including axon initial segments, cardiomyocyte T-tubules, and epithelial cell lateral membranes. Ankyrins are often co-expressed in the same cells and, although structurally similar, have non-overlapping functions. We previously determined that the regulatory domain of ankyrin-B defines specificity between ankyrins B and G in cardiomyocytes. Here, we identify key residues on the surface of an amphipathic alpha-helix unique to the regulatory domain of ankyrin-B that are essential for the function of ankyrin-B in cardiomyocytes. Using circular dichroism, we determined that a peptide representing the predicted helix folds as a helix in solution. Alanine-scanning mutagenesis revealed that residues 1773, 1777, 1780, 1784, and 1788 located in a patch on one surface the helix are critical for ankyrin-B function in cardiomyocytes. In a parallel set of experiments we determined that the molecular co-chaperone human DnaJ homologue 1 (Hdj1)/Hsp40 interacts with the ankyrin-B regulatory domain. Moreover, interaction of Hdj1/Hsp40 with the regulatory domain was mapped by random mutagenesis to same surface of the alpha-helix that is required for ankyrin-B function. These results provide new insight into the molecular basis for specificity between ankyrin-based pathways by defining a key alpha-helix structure in the divergent regulatory domain of ankyrin-B as well as interaction of the helix with Hdj1/Hsp40, the first downstream target for ankyrin-B-specific function.

Inositol 1,4,5-trisphosphate receptor localization and stability in neonatal cardiomyocytes requires interaction with ankyrin-B.

The molecular mechanisms required for inositol 1,4,5-trisphosphate receptor (InsP(3)R) targeting to specialized endoplasmic reticulum membrane domains are unknown. We report here a direct, high affinity interaction between InsP(3)R and ankyrin-B and demonstrate that this association is critical for InsP(3)R post-translational stability and localization in cultures of neonatal cardiomyocytes. Recombinant ankyrin-B membrane-binding domain directly interacts with purified cerebellar InsP(3)R (K(d) = 2 nm). 220-kDa ankyrin-B co-immunoprecipitates with InsP(3)R in tissue extracts from brain, heart, and lung. Alanine-scanning mutagenesis of the ankyrin-B ANK (ankyrin repeat) repeat beta-hairpin loop tips revealed that consecutive ANK repeat beta-hairpin loop tips (repeats 22-24) are required for InsP(3)R interaction, thus providing the first detailed evidence of how ankyrin polypeptides associate with membrane proteins. Pulse-chase biosynthesis experiments demonstrate that reduction or loss of ankyrin-B in ankyrin-B (+/-) or ankyrin-B (-/-) neonatal cardiomyocytes leads to approximately 3-fold reduction in half-life of newly synthesized InsP(3)R. Furthermore, interactions with ankyrin-B are required for InsP(3)R stability as abnormal InsP(3)R phenotypes, including mis-localization, and reduced half-life in ankyrin-B (+/-) cardiomyocytes can be rescued by green fluorescent protein (GFP)-220-kDa ankyrin-B but not by GFP-220-kDa ankyrin-B mutants, which do not associate with InsP(3)R. These new results provide the first physiological evidence of a molecular partner required for early post-translational stability of InsP(3)R.