Ezrin Controls the Macromolecular Complexes Formed between an Adapter Protein Na+/H+ Exchanger Regulatory Factor and the Cystic Fibrosis Transmembrane Conductance Regulator

Na+/H+ exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)2·NHERF·ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR·NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton. Na+/H+ exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)2·NHERF·ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR·NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton. Na+/H+exchanger regulator factor (NHERF) 2The abbreviations used are: NHERFNa+/H+ exchanger regulatory factorCFTRcystic fibrosis transmembrane conductance regulatorC-CFTRthe last 70 amino acid residues (1411-1480) at the carboxyl terminus of CFTRERMezrin-radixin-moesinFERMa conserved domain of ∼300 amino acid residues shared by a family proteins that include protein 4.1ezrinradixin, and moesinezFERMthe FERM domain of ezrinNHE3Na+/H+ exchanger isoform 3PDZa modular domain whose name is derived from the first three proteins (postsynaptic density 95, disk-large, zo-1) from which this domain is discoveredPDZ1The first amino-terminal PDZ domain of NHERF, amino acid residues 11-99PDZ2the second PDZ domains of NHERF, amino acid residues 150-240PDZ1-PDZ22fragment of NHERF containing the PDZ1 and PDZ2 domains, residues 11-240PDZ2-CTtruncated NHERF containing the PDZ2 domain and the C terminus, residues 150-358RUresponse unit in surface plasmon resonance experimentsSPRsurface plasmon resonanceSAXSsmall angle x-ray scatteringTBSTris-buffered saline is an adaptor protein that is responsible for organizing membrane channels and receptors (1Weinman E.J. 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Na+/H+ exchanger regulatory factor cystic fibrosis transmembrane conductance regulator the last 70 amino acid residues (1411-1480) at the carboxyl terminus of CFTR ezrin-radixin-moesin a conserved domain of ∼300 amino acid residues shared by a family proteins that include protein 4.1 radixin, and moesin the FERM domain of ezrin Na+/H+ exchanger isoform 3 a modular domain whose name is derived from the first three proteins (postsynaptic density 95, disk-large, zo-1) from which this domain is discovered The first amino-terminal PDZ domain of NHERF, amino acid residues 11-99 the second PDZ domains of NHERF, amino acid residues 150-240 fragment of NHERF containing the PDZ1 and PDZ2 domains, residues 11-240 truncated NHERF containing the PDZ2 domain and the C terminus, residues 150-358 response unit in surface plasmon resonance experiments surface plasmon resonance small angle x-ray scattering Tris-buffered saline NHERF is a multidomain and multivalent protein that recruits different signaling The amino terminus of NHERF contains two modular PDZ derived from the first three proteins that this domain was identified density 95/disk-large/zonula occluden-1) domains, PDZ1 and PDZ2 The NHERF PDZ domains bind to the amino acid at the carboxyl of a number of membrane channels or receptors S. J.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J.A. J. Mol. Biol. 2001; PubMed Scopus Google Scholar, S. J.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. J. Biol. 2003; PubMed Scopus (18) Google Scholar, C. J. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. J.G. A.M. M. 1998; 20: Full Text Full Text PDF PubMed Scopus Google Scholar). The carboxyl terminus of NHERF the FERM domain (a conserved domain that is shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, which as proteins NHERF is responsible for the interactions cell membrane and the a membrane adapter NHERF the and of membrane by S. J.W. Weinman E.J. 2004; PubMed Scopus Google Scholar). that the by NHERF form and cystic fibrosis transmembrane conductance regulator and membrane proteins to the of CFTR W.B. Am. J. 2004; PubMed Scopus Google Scholar, M.J. W.B. 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However, NHERF interacts with ezrin to the of membrane proteins is Here we a of and in studies to show that ezrin regulates the cooperative of CFTR cytoplasmic domains by to the of NHERF and ezrin in the apical membranes of polarized epithelial the cooperative of NHERF may be employed as a general mechanism to the and of membrane channels and receptors by the membrane cytoskeleton. and the full-length and ezrin from The the CFTR was by J. Foskett of We employed the and to proteins in the and the amino-terminal The a at the amino terminus of the proteins, the an amino-terminal in the The regions the amino acid of 11-99 150-240 11-240 150-358 of NHERF, the FERM domain of ezrin of ezrin and the last 70 amino acid residues (1411-1480) at the carboxyl terminus of CFTR by The in the of the in the cells large by and with of of containing and The proteins first by a with and with the binding and The proteins and by an with a The of the proteins, to amino acid for for for for for NHERF, for and for The protein by at the by the scattering experiments a with the of scattering and scattering experiments scattering the of the protein or protein in scattering the and the of the protein scattering the at for to of protein was to the for each The of is scattering experiments at plasmon resonance experiments The of a was by and the binding and the truncated of NHERF the the was with by 3 of at in the the binding of to NHERF binding to the C-CFTR·NHERF the was with by 3 of in the was and by The density was by the binding response from to response The NHERF, at a of the surface at for 3 The NHERF or C-CFTR·NHERF the The response by the from a cell with the of the to the The binding was by the from The binding an in The to the was as a to the binding The binding to the binding to or to the binding to or A.B. C. C. J. Mol. Biol. 2003; PubMed Scopus (76) Google Scholar). and in cells by L. for The cells in with at in a The cells in a with of The was used to NHERF or the in NHERF and the to saline The NHERF an amino-terminal and the an amino-terminal of the protein was to the containing the The was by the protein and for The at for was to the to a of the cell density the was from the by The cells with of was to each of the was to the The cell was at and in a for NHERF and in we first in cells NHERF in The cells for NHERF was Immunoprecipitation and in with saline by in at of was to of cell and the was in a for to of and the was in a The and three with of and in of of was to of cell to proteins in the to membranes by The membranes in in for 3 at The was used for for NHERF or for The was to the and in The the with for three and with the for and or for for at and for three with of The by for experiments with an with a from to is the of the scattering is the scattering and is the of the previously J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. 1998; Scopus Google Scholar). The scattering as scattering The of which is to the and of a be from the O. O. Scholar, of 1998; by in the The scattering is to the of the protein of the distribution which is the of two scattering at a from each in the by the J. Scopus Google Scholar). The at which the of the The of and the of be used to the and the two domains the The J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) was used to the from The J. 2001; Scopus Google Scholar) was used to the of and to the the for of the from previously Y. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). an adapter NHERF the of protein is of to the and the of the NHERF to NHERF a for protein binding partners in and scattering experiments show that the of NHERF protein of the of NHERF is However, the protein is to and scattering experiments show that NHERF as a with a of and we show in the is the of NHERF that is responsible for with the cytoplasmic tail of CFTR and the FERM domain of NHERF is the form in this The NHERF and an to NHERF from binding to of and NHERF and the by scattering and of the was from the by in by by from at at at at at at at from and as shown in from and as shown in The of the was from the by The from The from and as shown in in a The and binding of to the NHERF by a of and NHERF was with at different the was by The with the of to NHERF at 1:1 and shown in of only which is at scattering experiments show that the and a of which the of a 1:1 C-CFTR·NHERF complex. experiments to the and of NHERF to the to NHERF binding from which be by the binding A.B. C. C. J. Mol. Biol. 2003; PubMed Scopus (76) Google Scholar) with two different and We a of experiments to the binding of PDZ different the two and the PDZ1 domain from The in The binding of PDZ1 for is that of PDZ2 PDZ2 and binding for for and for a binding to that of of the binding by The from the of the response unit of the by into or was to the binding of the PDZ1 domain of NHERF, and to that of in a the of C-CFTR·NHERF binding with of the truncated NHERF we to the C-CFTR·NHERF as binding, which is and as binding, which is that the PDZ1 domain in the of and in the full-length NHERF show in binding for with that of the PDZ1 that the binding of PDZ1 to is and by studies identified the NHERF and the FERM domain of ezrin D. A. J. Biol. 2001; PubMed Scopus Google Scholar). We the and the of NHERF binding to by and was with different of NHERF the at a of in the the of the of NHERF with by a scattering experiments the show that the form and a of The of the by scattering is that NHERF and form a 1:1 complex. The of the was by the binding in experiments with a of experiments show a and specific 1:1 binding NHERF and of and was with the at the be identified and by of of with two and the at of and scattering experiments show that the of two the to be and The and scattering that the two of ternary the binding of as The different of the two a in and of two The at is a ternary with to the first PDZ domain of the that at is the with binding to the second PDZ is the binding of at the PDZ1 domain of the ternary to be and to a the ternary with binding to the PDZ2 However, the of the two by scattering with and a of the two scattering experiments that the PDZ2 domain of NHERF in the an binding to the cytoplasmic tails of as with that in the NHERF of the 2:1:1 We with the at of this a The of this at that a is which is the scattering of this that this ternary with a the of a and The by scattering is a 2:1:1 binding and the of ternary complex. The that the to binding is with the binding with two and and that the binding of for the is different from that of to NHERF binding, in of the of the and the of the of the binding the of to binding by with the of to NHERF the of the and is that of to NHERF binding the two binding the as a of binding to NHERF, NHERF of with with the of a 2:1:1 a 1:1 C-CFTR·NHERF complex. we which PDZ domain of NHERF an binding for the FERM domain of ezrin is to the NHERF carboxyl We the binding of to truncated of that the of PDZ1 to binding is to of to The of PDZ1 is to the of NHERF and that of complex. the of PDZ1 in different and in different that the PDZ1 domain of NHERF an binding for and that binding is by ezrin binding to NHERF, or by the of the PDZ2 domain of PDZ2 binding we the binding of a truncated with the second PDZ domain and the carboxyl terminus Without binding of at the carboxyl terminus of the of for is with a and The binding of for is as as that of the PDZ2 domain for However, the carboxyl terminus of is to the for is with a and scattering experiments that form a 1:1 The and scattering experiments that is the PDZ2 domain of NHERF that an binding to the FERM domain of ezrin is to the carboxyl terminus of the show that binding of the FERM domain of ezrin at the NHERF carboxyl terminus the of to NHERF We that is the PDZ2 domain of NHERF that an binding for the binding of PDZ1 by ezrin Immunoprecipitation and the of NHERF with CFTR and with the FERM of in and immunoblotting experiments to the interactions of NHERF with and with the full-length CFTR in cells by full-length CFTR M. M. D. N. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. M. L. M. B. Grinstein S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the immunoblotting of CFTR from the cell We first the of NHERF with CFTR in the NHERF an amino-terminal was in cells for the cells was used to NHERF in that the contains the full-length CFTR and NHERF of The and NHERF in cells to the interactions of NHERF with CFTR and with in The contains an amino-terminal was used to the NHERF that the contains NHERF, and 3 of to that shown in by to the and immunoblotting show that NHERF interacts with the full-length CFTR and with the FERM domain of ezrin to form a ternary in vivo. The of the that we in and 3 of the A of the of CFTR in and 3 that the bind CFTR in that in that an binding to However, we that the we in this to the binding and of the experiments Mol. Biol. 2004; Google Scholar). of to the PDZ of NHERF the of the of NHERF to the FERM of was used to the binding of the FERM domain of ezrin to the C-CFTR·NHERF complex. of and NHERF, the was by The C-CFTR·NHERF at was an The shown in that the binding the FERM domain of ezrin and the C-CFTR·NHERF is with a in which is that of to NHERF binding the of The NHERF in the C-CFTR·NHERF an binding for NHERF The of the the of the 2:1:1 ternary by The a of The an with the for the 2:1:1 The of the ternary from is shown in we the two the two to the PDZ1 and PDZ2 domains of the of the of the ternary the be that the the two is in a that is to the of of a CFTR channel from a S. M. Proc. Natl. Acad. Sci. U. S. 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Biol. 2002; PubMed Scopus Google Scholar). we a of of ezrin by ezrin to NHERF The subsequent of PDZ2 NHERF to bring two CFTR carboxyl into proximity to each and as a to a CFTR the regulatory be from the in family this mechanism to interact with NHERF in the regulatory from the to transmembrane we shown that the of in the PDZ1 domain of NHERF the of the NHERF carboxyl terminus for by The in binding that binding of to the PDZ1 domain of NHERF the of NHERF with The that the may a in from CFTR to the the of is the is from the to ezrin and to the CFTR this to the binding of CFTR to the PDZ1 domain of NHERF, as a to the of the PDZ2 domain with by ezrin binding to a the of CFTR channel to the of NHERF by ezrin is the 2:1:1 is which may that the binding of to the PDZ2 domain of NHERF the the NHERF carboxyl terminus and the NHERF binding may the of NHERF binding to NHERF proteins that the PDZ domain binding regulation of the of the transmembrane transporter Na+/H+ exchanger 3 of which NHERF is an essential ezrin an for E.J. 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