Ankyrin-G and β2-Spectrin Collaborate in Biogenesis of Lateral Membrane of Human Bronchial Epithelial Cells

Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of β2-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and β2-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells. Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of β2-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and β2-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells. Physiological functions of most membrane-spanning proteins of metazoans require that the proteins localize within specialized domains within the plasma membrane or endoplasmic reticulum. Emerging evidence from animal models and human mutations suggests that one of the mechanisms for organizing functionally related subsets of proteins in specialized membrane domains operates through the ankyrin family of membrane adapters (1Mohler P.J. Bennett V. Front. Biosci. 2005; 10: 2832-2840Crossref PubMed Scopus (25) Google Scholar). Ankyrin-G is required for neurons to be able to fire action potentials and for the clustering of Nav1.6, KCNQ2/3 channels, and the L1 cell adhesion molecule neurofascin at axon initial segments (2Ango F. de Cristo G. Higashiyama H. Bennett V. Wu P. Huang Z.J. Cell. 2004; 119: 257-272Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 3Jenkins S.M. Bennett V. J. Cell Biol. 2001; 155: 739-746Crossref PubMed Scopus (352) Google Scholar, 4Pan Z. Kao T. Horvath Z. Lemos J. Sul J.Y. Cranstoun S.D. Bennett V. Scherer S.S. Cooper E.C. J. Neurosci. 2006; 26: 2599-2613Crossref PubMed Scopus (422) Google Scholar, 5Zhou D. Lambert S. Malen P.L. Carpenter S. Boland L.M. Bennett V. J. Cell Biol. 1998; 143: 1295-1304Crossref PubMed Scopus (461) Google Scholar). A human mutation in the ankyrin-G-binding site of Nav1.5 blocks its clustering at intercalated discs and transverse tubule sites in cardiomyocytes and causes a cardiac arrhythmia (6Mohler P.J. Rivolta I. Napolitano C. LeMaillet G. Lambert S. Priori S.G. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17533-17538Crossref PubMed Scopus (309) Google Scholar). Moreover, ankyrin-G is required for targeting the RhBG ammonium transporter to basolateral domains of kidney epithelial cells (7Lopez C. Metral S. Eladari D. Drevensek S. Gane P. Chambrey R. Bennett V. Cartron J.P. Le Van Kim C. Colin Y. J. Biol. Chem. 2005; 280: 8221-8228Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Ankyrin-B is required for localization of InsP 3The abbreviations used are: InsP3, inositol trisphosphate; HBE, human bronchial epithelial; GFP, green fluorescent protein; siRNA, small interfering RNA; HA, hemagglutinin; HEK, human embryonic kidney. 3The abbreviations used are: InsP3, inositol trisphosphate; HBE, human bronchial epithelial; GFP, green fluorescent protein; siRNA, small interfering RNA; HA, hemagglutinin; HEK, human embryonic kidney.3 receptors at specialized sites within the sarcoplasmic reticulum of cardiomyocytes (8Mohler P.J. Davis J.Q. Davis L.H. Hoffman J.A. Michaely P. Bennett V. J. Biol. Chem. 2004; 279: 12980-12987Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Mohler P.J. Gramolini A.O. Bennett V. J. Biol. Chem. 2002; 277: 10599-10607Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 10Mohler P.J. Schott J.J. Gramolini A.O. Dilly K.W. Guatimosim S. duBell W.H. Song L.S. Haurogne K. Kyndt F. Ali M.E. Rogers T.B. Lederer W.J. Escande D. Le Marec H. Bennett V. Nature. 2003; 421: 634-639Crossref PubMed Scopus (831) Google Scholar, 11Tuvia S. Buhusi M. Davis L. Reedy M. Bennett V. J. Cell Biol. 1999; 147: 995-1008Crossref PubMed Scopus (109) Google Scholar). Ankyrin-B-dependent clusters of InsP3 receptors in the sarcoplasmic reticulum are coincident with Na/K-ATPase and Na+/Ca2+ exchanger on the transverse tubules and are a specialized feature of cardiomyocytes not found in skeletal muscle (12Mohler P.J. Davis J.Q. Bennett V. PLoS Biol. 2005; 3: e423Crossref PubMed Scopus (198) Google Scholar). Ankyrin-B mutations that block the ability to segregate InsP3 receptors Na/K-ATPase and Na+/Ca2+ exchanger cause a dominantly inherited fatal cardiac arrhythmia (10Mohler P.J. Schott J.J. Gramolini A.O. Dilly K.W. Guatimosim S. duBell W.H. Song L.S. Haurogne K. Kyndt F. Ali M.E. Rogers T.B. Lederer W.J. Escande D. Le Marec H. Bennett V. Nature. 2003; 421: 634-639Crossref PubMed Scopus (831) Google Scholar, 12Mohler P.J. Davis J.Q. Bennett V. PLoS Biol. 2005; 3: e423Crossref PubMed Scopus (198) Google Scholar, 13Mohler P.J. Splawski I. Napolitano C. Bottelli G. Sharpe L. Timothy K. Priori S.G. Keating M.T. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9137-9142Crossref PubMed Scopus (276) Google Scholar). Mechanisms for ankyrin-based targeting of membrane proteins have been explored in cultured epithelial cells (14Nelson W.J. Shore E.M. Wang A.Z. Hammerton R.W. J. Cell Biol. 1990; 110: 349-357Crossref PubMed Scopus (244) Google Scholar, 15Nelson W.J. Veshnock P.J. Nature. 1987; 328: 533-536Crossref PubMed Scopus (340) Google Scholar, 16Yeaman C. Grindstaff K.K. Nelson W.J. Physiol. Rev. 1999; 79: 73-98Crossref PubMed Scopus (444) Google Scholar). Early studies led to a proposal of an outside-in hierarchy of protein interactions where homotypic adhesion of E-cadherin at sites of cell-cell contact results in recruitment of spectrin followed by ankyrin and its partner, the Na/K-ATPase (16Yeaman C. Grindstaff K.K. Nelson W.J. Physiol. Rev. 1999; 79: 73-98Crossref PubMed Scopus (444) Google Scholar). Recent siRNA-based methods for inhibiting protein expression have allowed direct tests of the role of ankyrin in membrane protein organization. A surprising result of elimination of 190-kDa ankyrin-G was that columnar epithelial cells no longer formed a lateral membrane and developed a squamous morphology (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These results suggest that ankyrin-G directly participates in de novo biogenesis of the lateral membrane. Ankyrin-B and ankyrin-G are overall quite similar in amino acid sequence, with the major differences primarily residing in their C-terminal regulatory domains (9Mohler P.J. Gramolini A.O. Bennett V. J. Biol. Chem. 2002; 277: 10599-10607Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18Abdi K.M. Mohler P.J. Davis J.Q. Bennett V. J. Biol. Chem. 2006; 281: 5741-5749Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 19Mohler P.J. Hoffman J.A. Davis J.Q. Abdi K.M. Kim C.R. Jones S.K. Davis L.H. Roberts K.F. Bennett V. J. Biol. Chem. 2004; 279: 25798-25804Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The high level of sequence similarity in membrane-binding and spectrin-binding domains of ankyrin-B and -G suggests the possibility of conserved mechanisms underlying their apparently distinct functions. In this study, we have further compared the spectrin-binding domains of ankyrin-B and ankyrin-G by performing systematic alanine-scanning mutagenesis to determine functionally important amino acid residues predicted to be accessible to solvent. Such surface-exposed residues would be available for protein-protein interactions. We identified two homologous sites that are not required for spectrin binding or cellular targeting, but are essential for function of both ankyrins-B and -G. We have previously reported that ankyrin-B does not require β2-spectrin for localization of InsP3 receptors, although ankyrin-B is required for cellular targeting of β2-spectrin in cardiomyocytes (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). We demonstrate here that in contrast to ankyrin-B, ankyrin-G requires β2-spectrin for its function in epithelial cells. Ankyrin Mutagenesis—Clusters of charged residues predicted to be surface exposed were mutated to alanines in the spectrin-binding domain of human 220-kDa ankyrin-B and rat 190-kDa ankyrin-G. The A1024P mutant, which is comparable with the spectrin binding-defective A1000P mutation in ankyrin-B (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), was introduced into the ankyrin-G spectrinbinding domain. All mutations were introduced using the QuikChange XL site-directed mutagenesis kit (Stratagene). DNA sequencing confirmed all the alanine mutations were present with no additional mutation in the spectrin-binding domain. The plasmids human ankyrin-B and rat ankyrin-G have been (9Mohler P.J. Gramolini A.O. Bennett V. J. Biol. Chem. 2002; 277: 10599-10607Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Ankyrin-B mutants and Ankyrin-G mutants The of mutation the and the mutated and was into human bronchial epithelial cells using the by R. R. 2002; PubMed Scopus Google Scholar). The used to human 190-kDa ankyrin-G been in an (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The rat 190-kDa ankyrin-G is to the targeting the human 190-kDa ankyrin-G of and be used to the ankyrin-G in cells (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The in the human β2-spectrin was on by R. R. 2002; PubMed Scopus Google Scholar). A corresponding was generated by mutations at in the human β2-spectrin to to be used a β2-spectrin a C-terminal of human β2-spectrin was from β2-spectrin M. Bennett V. J. Biol. Chem. Full Text PDF PubMed Google using a β2-spectrin for the sequence on the was mutated at were mutated in the for human to to the human β2-spectrin and cardiomyocytes were from the of and previously S. Buhusi M. Davis L. Reedy M. Bennett V. J. Cell Biol. 1999; 147: 995-1008Crossref PubMed Scopus (109) Google Scholar, P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). bronchial epithelial cells were cultured and (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). the cells were and in the cells for a to cells were on the of a cells were in a from for to proteins that to and to the of cell The in a were with of ankyrin-G or β2-spectrin and of using The of plasmids that cells the also be with or a for cells that have been with the also results in a of cells with These cells an for in cells were by membrane was by the of the by from the of the cell in an cells were with ankyrin-B and ankyrin-G mutants using Cell were to and with was used to β2-spectrin were with cells at cells and with of β2-spectrin and were into cells Cell were with β2-spectrin were using protein (9Mohler P.J. Gramolini A.O. Bennett V. J. Biol. Chem. 2002; 277: 10599-10607Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). were and with previously (10Mohler P.J. Schott J.J. Gramolini A.O. Dilly K.W. Guatimosim S. duBell W.H. Song L.S. Haurogne K. Kyndt F. Ali M.E. Rogers T.B. Lederer W.J. Escande D. Le Marec H. Bennett V. Nature. 2003; 421: 634-639Crossref PubMed Scopus (831) Google Scholar, K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). cardiomyocytes were with and and with and in bronchial epithelial cells were with and with and in the cells were with at and with and for at The ankyrin-B, InsP3 and were (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). and Na/K-ATPase were The was a of G. were with a using either a or a for of the spectrin-binding domain of ankyrin-B with β2-spectrin been (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The spectrin-binding domain of rat 190-kDa ankyrin-G was and the and sites of The spectrin-binding domain alanine mutants from the ankyrin-B and the ankyrin-G mutants were into using the and domain mutants were with the β2-spectrin into and for a using and of in the of Ankyrin-B and residues on the surface of the spectrin-binding we generated a of mutations in and where charged clusters of two or residues were with The of using charged residues to surface residues is in a of where of predicted surface residues were in on the on the K.F. D. PubMed Google Scholar). mutants were generated in ankyrin-B, and mutants were generated in ankyrin-G DNA sequencing confirmed all the alanine mutations additional mutation in cause a in protein and loss of the alanine mutants were in cells and by with in all mutants of ankyrin-B and -G that resulted in loss of function generated in cells. In all ankyrin-G mutants were to the lateral membrane where ankyrin-G is in human bronchial epithelial cells (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google the of all ankyrin-B mutants were and in cardiomyocytes and These results that the mutant are major mutants are and exhibit cellular A and of of cells either or mutants with comparable with corresponding of mutant the amino acid and the amino the residues in the Ankyrin-B and ankyrin-G mutants are that loss of function and ankyrin-G mutants to the lateral membrane in human bronchial epithelial cells in All ankyrin-G mutants were also to the lateral of ankyrin-B mutants in mouse cardiomyocytes a of ankyrin-B and InsP3 receptor of ankyrin-B in cardiomyocytes results in abnormal localization of InsP3 localization of mutants and InsP3 receptor in mouse and to InsP3 receptor localization a of the of the ankyrin-B the abnormal InsP3 receptor localization in of alanine mutants of ankyrin-B were evaluated to determine mutant was able to abnormal InsP3 receptor localization in mouse the mutants we identified that were to the InsP3 receptor localization in cardiomyocytes These mutants and was a mutant in this (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). All ankyrin-B mutants InsP3 receptor of Interestingly, the mutants are in and are within the spectrin-binding domain to (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google and and and are to the site of the human mutation which causes (10Mohler P.J. Schott J.J. Gramolini A.O. Dilly K.W. Guatimosim S. duBell W.H. Song L.S. Haurogne K. Kyndt F. Ali M.E. Rogers T.B. Lederer W.J. Escande D. Le Marec H. Bennett V. Nature. 2003; 421: 634-639Crossref PubMed Scopus (831) Google Scholar). of ankyrin-G mutants were in human bronchial epithelial cells where of 190-kDa ankyrin-G by RNA causes a loss of lateral membrane and an of columnar cells to a squamous morphology of rat which different within the human sequence and is to human siRNA, loss of lateral membrane (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). and mutants of and to the lateral membrane the depletion of ankyrin-G of a was to the of the lateral membrane. was used to cells with both the ankyrin-G and the Interestingly, the ankyrin-G mutant was in targeting and the corresponding ankyrin-B mutant was in suggests that the ankyrin-B mutation does not cause and Ankyrin-B and Ankyrin-G to sites of the spectrin-binding domain are ankyrin-B and -G These common sites are and of These sites are required for a that is not directly related to spectrin mutations did not spectrin in In these sites are conserved not in human ankyrin but also in of and In contrast to common mutants were and of ankyrin-B are specific to InsP3 receptor targeting function in cardiomyocytes, of ankyrin-G is specific to lateral membrane biogenesis in epithelial most of these residues are conserved ankyrin-B and -G. of of spectrin binding activity of The spectrin-binding domains from the mutants of ankyrin-B and ankyrin-G were into a and for β2-spectrin in of the spectrin-binding domain with β2-spectrin is by of on We previously that ankyrin-B mutants spectrin binding activity also not localization of β2-spectrin in cardiomyocytes, the in of the (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The is not required for InsP3 receptor targeting, the mutants activity InsP3 receptor with this all the mutants of spectrin binding activity in the ankyrin-G mutant, spectrin binding activity and the spectrin Ankyrin-B mutant corresponding to mutation of ankyrin-G at did not loss of function in cardiomyocytes (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google but did loss of spectrin binding activity We previously reported that A1000P mutation of ankyrin-B, identified in a for mutations affecting spectrin spectrin binding activity (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). A1000P mutation of ankyrin-B the ability to direct β2-spectrin targeting in cardiomyocytes but had no effect on the ability of ankyrin-B to direct the targeting of InsP3 receptors (20Mohler P.J. Yoon W. Bennett V. J. Biol. Chem. 2004; 279: 40185-40193Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). We evaluated the of the corresponding A1024P mutation in ankyrin-G on spectrin binding and activity in restoring lateral membrane the A1024P mutant ankyrin-G the ability to spectrin in the in contrast to ankyrin-B, the A1024P mutation of ankyrin-G also function in restoring lateral membrane in epithelial cells. The A1024P mutant ankyrin-G activity in localization to the lateral membrane and was a these results suggest that ankyrin-B, is by mutations at two distinct sites that result in loss of spectrin binding for in and determine is required for of the lateral we used to the protein in cells by of cell and by Cell was on but of β2-spectrin results in of the of the lateral membrane from to by a in the of and B and the in and were in the of the lateral membrane was The results from a of loss of lateral membrane and the to additional lateral membrane. is by depletion of β2-spectrin on the its localization R. J. Cell Biol. PubMed Scopus Google Scholar). The Na/K-ATPase was from the lateral membrane but did not to the membrane was present at the of the and lateral Moreover, E-cadherin was to the lateral surface mutated at sites in the targeting sequence, was to and loss of the lateral membrane of β2-spectrin the of 190-kDa with loss of lateral of and membrane and of result is with that expression of domains of β2-spectrin morphology in cultured epithelial cells S. Bennett V. J. Cell Biol. PubMed Scopus Google and that is required for of Na/K-ATPase in epithelial cells Wang P. S. J. L.S. J. Cell Biol. PubMed Scopus Google Scholar). The of for in epithelial of C. M. Davis E.M. J. Cell Biol. PubMed Scopus Google Scholar, S. L. Bennett V. J. Cell Biol. PubMed Scopus Google differences and or the that most epithelial cells in this not have lateral lateral membrane is in epithelial cells and the role of β2-spectrin in this of membrane we the of protein on the of lateral membrane in identified by the a lateral membrane with β2-spectrin and In cells depleted of β2-spectrin that of the lateral which was to a of β2-spectrin siRNA, of the cells were in contrast to of cells in cells The of cells depletion of β2-spectrin is with of a failure in the of the lateral membrane. of 190-kDa ankyrin-G causes an in epithelial cells and also results in cells (17Kizhatil K. Bennett V. J. Biol. 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These sites also be for the to the site is conserved in all be important in to and protein that with these conserved of evidence that ankyrin-G and β2-spectrin in biogenesis of the lateral membrane of epithelial cells. and A1024P mutations that abolish spectrin binding in also abolish function in restoration of the lateral membrane in epithelial cells. Moreover, of β2-spectrin the depletion of ankyrin-G in interphase with loss of the lateral of and membrane and of cells from columnar to squamous In of β2-spectrin also the of new lateral membrane previously reported for cells (17Kizhatil K. Bennett V. J. Biol. Chem. 2004; 279: 16706-16714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The of lateral membrane biogenesis in cells with in where new lateral membrane is formed in the of T. J. Cell Biol. PubMed Scopus Google Scholar). 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