Key points are not available for this paper at this time.
Cyclic AMP is a major second messenger that inhibits the brush border Na+/H+exchanger NHE3. We have previously shown that either of two related regulatory proteins, E3KARP or NHERF, is necessary for the cAMP-dependent inhibition of NHE3. In the present study, we characterized the interaction between NHE3 and E3KARP using in vitro binding assays. We found that NHE3 directly binds to E3KARP and that the entirety of the second PSD-95/Dlg/ZO-1 (PDZ) domain plus the carboxyl-terminal domain of E3KARP are required to bind NHE3. E3KARP binds an internal region within the NHE3 C-terminal cytoplasmic tail, defining a new mode of PDZ domain interaction. Analyses of cellular distribution of NHE3 and E3KARP expressed in PS120 fibroblasts show that NHE3 and E3KARP are co-localized on the plasma membrane, but not in a distinct juxtanuclear compartment in which NHE3 is predominantly expressed. The distributions of NHE3 and E3KARP were not affected by treatment with 8-bromo-cAMP. As shown earlier for the human homolog of NHERF, we also found that the cytoskeletal protein ezrin binds to the carboxyl-terminal domain of E3KARP. These results are consistent with the possibility that E3KARP and NHERF may function as scaffold proteins that bind to both NHE3 and ezrin. Since ezrin is a protein kinase A anchoring protein, we suggest that the scaffolding function of E3KARP binding to both ezrin and NHE3 localizes cAMP-dependent protein kinase in the vicinity of the cytoplasmic domain of NHE3, which is phosphorylated by elevated cAMP. Cyclic AMP is a major second messenger that inhibits the brush border Na+/H+exchanger NHE3. We have previously shown that either of two related regulatory proteins, E3KARP or NHERF, is necessary for the cAMP-dependent inhibition of NHE3. In the present study, we characterized the interaction between NHE3 and E3KARP using in vitro binding assays. We found that NHE3 directly binds to E3KARP and that the entirety of the second PSD-95/Dlg/ZO-1 (PDZ) domain plus the carboxyl-terminal domain of E3KARP are required to bind NHE3. E3KARP binds an internal region within the NHE3 C-terminal cytoplasmic tail, defining a new mode of PDZ domain interaction. Analyses of cellular distribution of NHE3 and E3KARP expressed in PS120 fibroblasts show that NHE3 and E3KARP are co-localized on the plasma membrane, but not in a distinct juxtanuclear compartment in which NHE3 is predominantly expressed. The distributions of NHE3 and E3KARP were not affected by treatment with 8-bromo-cAMP. As shown earlier for the human homolog of NHERF, we also found that the cytoskeletal protein ezrin binds to the carboxyl-terminal domain of E3KARP. These results are consistent with the possibility that E3KARP and NHERF may function as scaffold proteins that bind to both NHE3 and ezrin. Since ezrin is a protein kinase A anchoring protein, we suggest that the scaffolding function of E3KARP binding to both ezrin and NHE3 localizes cAMP-dependent protein kinase in the vicinity of the cytoplasmic domain of NHE3, which is phosphorylated by elevated cAMP. cAMP-dependent protein kinase amino acid(s) polymerase chain reaction nitrilotriacetic acid maltose-binding protein N-terminal 302 aa of human ezrin 8-bromoadenosine 3′:5′-monophosphate phosphate-buffered saline PSD-95/Dlg/ZO-1. NHE3 is the Na+/H+ exchanger in the brush border membrane of the proximal tubule and small intestine and colon that plays a major role in transepithelial Na+ absorption (1Aronson P.S. Igarashi P. Curr. Top. Membr. Transp. 1986; 26: 57-75Crossref Scopus (21) Google Scholar, 2Yun C.H.C. Tse C.-M. Nath S.K. Levine S.A. Brant S.R. Donowitz M. Am. J. Physiol. 1995; 269: G1-G11Crossref PubMed Scopus (38) Google Scholar). Among the various stimuli that modulate NHE3 activity (1Aronson P.S. Igarashi P. Curr. Top. Membr. Transp. 1986; 26: 57-75Crossref Scopus (21) Google Scholar), cAMP inhibits the transepithelial Na+ absorption by NHE3. However, the molecular mechanisms of the cAMP-dependent inhibition of the Na+ absorptive process remain unknown. Currently, two major mechanisms have been suggested for the cAMP-dependent inhibition of NHE3 activity. The first involves an increase in NHE3 phosphorylation level by PKA,1 and the second requires the presence of a regulatory factor. NHE3 phosphorylation by PKA was initially demonstrated by Moe et al. (3Moe O.W. Amemiya M. Yamaji Y. J. Clin. Invest. 1995; 96: 2187-2194Crossref PubMed Scopus (89) Google Scholar), who showed that in AP-1 cells, a Chinese hamster ovary cell line, the cAMP-elicited inhibition of NHE3 activity was accompanied by an increase in NHE3 phosphorylation level. More recently, Kurashima et al. (4Kurashima K., Yu, F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) identified the sites of phosphorylation in NHE3 by PKA in AP-1 cells. In contrast, we previously reconstituted the cAMP-dependent inhibition of NHE3 by transfecting one of two regulatory proteins, E3KARP (NHE3kinase Aregulatoryprotein 2The corrected sequence is found under accession number AF004900 in the GenBankTM Data Bank. ; also named TKA-1) or NHERF (NHEregulatory factor), in the PS120 fibroblast cell line, which lacks these regulatory proteins, and consequently, cAMP does not affect NHE3 activity (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar, 6Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar, 7Weinman E.J. Steplock D. Tate K. Hall R.A. Spurney R.F. Shenolikar S. J. Clin. Invest. 1998; 101: 2199-2206Crossref PubMed Scopus (89) Google Scholar). This demonstrates the necessity of a regulatory protein in cAMP-dependent inhibition of NHE3. E3KARP was cloned from a human lung library by yeast two-hybrid screening using the cytoplasmic domain of NHE3 as bait (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). NHERF was identified by limited trypsinization and cellular fractionation of rabbit renal brush border vesicles (6Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar, 8Weinman E.J. Steplock D. Shenolikar S. J. Clin. Invest. 1993; 92: 1781-1786Crossref PubMed Scopus (118) Google Scholar). E3KARP and NHERF are closely related proteins of 337 and 358 aa, respectively. These proteins share 52% identity, and both contain two tandem protein interaction module PDZ domains (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). How these regulatory proteins mediate the inhibitory effect of cAMP is not clear. Protein-protein interactions are intrinsic to virtually every cellular process. Many of these interactions are mediated by modular domains such as the Src homology SH2 and SH3, pleckstrin homology, and PDZ domains (9Couture C. Deckert M. Williams S. Russo F.O. Altman A. Mustelin T. J. Biol. Chem. 1996; 271: 24294-24299Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 10Ferguson K.M. Lemmon M.A. Schlessinger J. Singler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 11Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (897) Google Scholar, 12Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1628) Google Scholar). We have shown previously that NHE3 and E3KARP directly interact with each other (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). In this present report, we analyzed the interaction between NHE3 and E3KARP to determine minimal domains necessary for the interaction. We found that the interaction of NHE3 with E3KARP represents a new mode of PDZ binding and that it requires a PDZ domain and a non-PDZ domain of E3KARP. We also found that E3KARP binds to an internal domain within the NHE3 cytoplasmic tail, which has previously been shown to be essential for PKA-dependent inhibition of NHE3 (13Cabado A.G., Yu, F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In addition, we show that E3KARP binds to the cytoskeletal protein ezrin, which was previously shown to bind to PKA. These findings suggest a potential model for E3KARP in the inhibition of NHE3 activity by PKA. PS120 fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 25 mmNaHCO3, 10 mm HEPES, 50 IU/ml penicillin, 50 μg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2, 95% air incubator at 37 °C. Where appropriate, G418 (800 μg/ml) or hygromycin (600 units/ml) was used to maintain selection pressure. Various domains of E3KARP were generated by PCR and were expressed as recombinant proteins in Escherichia coli. These domains include the first PDZ domain (P1) and the second PDZ domain (P2), expressed individually (P1 or P2) or together as a unit (P1–2); the C-terminal domain (C); and the second PDZ domain and the C-terminal domain as a single unit (P2-C) (Fig. 1). We also expressed clone 42 (C42), which extends from aa 130 to 314 of E3KARP and was one of the partial clones initially obtained from yeast two-hybrid screening (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). Fidelity of the PCR products was confirmed by nucleotide sequencing. The PCR products were cloned into pET30a (Novagen), expressed as hexahistidine (His6)-tagged fusion proteins in E. coli, and affinity-purified with Ni2+-nitrilotriacetic acid (NTA) resin as suggested by the manufacturer (QIAGEN Inc.). The above constructs were also expressed as maltose-binding protein (MBP) fusion proteins by cloning into pMAL-c2 (New England Biolabs Inc.). The entire rabbit NHE3 carboxyl-terminal construct extends from aa 475 to 832 (C3-832), and NHE3 C-terminal truncation constructs terminating at aa 711 (C3-711), 660 (C3-660), and 585 (C3-585) were generated by PCR and cloned into pET30a. The C-terminal 88 aa of NHE3 were generated by the NHE3 with by with with and into with and was in rabbit the carboxyl-terminal aa of E3KARP expressed as was by by E3KARP was initially cloned into pET30a The entire construct of the N-terminal and in with E3KARP was into in fibroblasts were with in E3KARP and was with and of was by is rabbit NHE3 at with a as previously (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). PS120 fibroblasts were in mm mm μg/ml and by at at for The was with were by binding to protein resin and were with to The presence of recombinant was with as by The presence of was with J. 1986; PubMed Scopus Google Scholar). The entire NHE3 and the above were with using the in to the of the in vitro products were by the products on by of E3KARP were generated as of each recombinant protein was by and were by for in 5% 25 mm and mm and were with of the entire NHE3 or NHE3 in 5% 25 mm mm and at °C. were with 25 mm and and NHE3 was by The N-terminal 302 aa of human ezrin were generated by and the PCR was into pET30a. The was with by in vitro and was used in as fibroblasts were in with mm The was at at for and the was with 10 of recombinant E3KARP or various domains of E3KARP on at °C. The were with and with by in The were by and were with the PS120 fibroblasts were grown on were and were with mm 8-bromoadenosine 3′:5′-monophosphate for with with were at for 10 with in by with in for 10 at were in and bovine serum for at were for at in 10% and bovine serum at the for for and for were with bovine serum and and for with were with bovine serum and and with Inc.). were with Inc.). were with an to a unit The Na+/H+ exchanger of PS120 were using the as previously S.A. Tse C.-M. Donowitz M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). was were with for the and the that E3KARP and NHE3 interact in we expressed E3KARP as a and fusion protein, in that the of E3KARP protein with and not function in cAMP-dependent inhibition of NHE3, we the activity of in to that the to activity is in the presence of in cells. The of in the or presence of was and by We was interaction between and in The the C-terminal aa of expressed in fibroblasts (Fig. The C-terminal aa of E3KARP used in of show limited homology to the C-terminal domain of NHERF (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). minimal with NHERF (Fig. was with and the of was with (Fig. was with the As shown in with the interaction in between NHE3 and consistent with earlier (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). determine which of E3KARP interact with the NHE3 we expressed and the entire E3KARP and various of E3KARP as recombinant The entire NHE3 with by in was used as a As shown in to the E3KARP protein, but not to PDZ domains expressed individually (P1 or P2) or as one unit We binding of to the and but this interaction not The not bind to the C-terminal domain of but the and C-terminal domains were expressed as one unit to the domain also to the consistent with the interaction of clone with in the yeast two-hybrid (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). the above we a on from fibroblasts using recombinant E3KARP and on The presence of to the recombinant proteins was analyzed by using the the As shown in to E3KARP and the and but not bind to the and C-terminal domains or to the of the binding of to the we expressed the domains of E3KARP as fusion These were with As shown in to and but not or the results shown in A. the of interaction was by the with and expressed as proteins μg/ml) as that in the presence of protein, binding was the domain binding to E3KARP and the In contrast, the domain showed effect on the consistent with the binding in the We which of the NHE3 with E3KARP. were on E3KARP using NHE3 C-terminal As shown in the entire NHE3 and which from aa 475 to to E3KARP. to aa to the binding of to E3KARP was that of However, not interact with E3KARP. PDZ domains interact with the of protein H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1628) Google Scholar, G. M. 1997; PubMed Scopus Google Scholar), the carboxyl-terminal 88 aa of NHE3 were expressed and used as a for a to determine this of NHE3 to the interaction with E3KARP. However, not interact with E3KARP. This of binding of the C-terminal 88 aa of NHE3 to the regulatory proteins is consistent with the that E3KARP with the of NHE3 is by a (Fig. We the distribution of E3KARP and expressed in PS120 We first the distribution of E3KARP in PS120 fibroblasts in the of NHE3. PS120 fibroblasts were with and by μg/ml were and with for to A that E3KARP is the with at or the plasma with not affect the distribution of E3KARP. In to a that a of was present in P. M. S. P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), E3KARP was not present in the of E3KARP not with that of which were with E3KARP distribution was also in the presence of that showed in juxtanuclear consistent with an earlier that NHE3 is in vesicles S. Yu, K. Lukacs G. K. Orlowski J. Grinstein S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In to the juxtanuclear of at or the plasma membrane was In contrast, E3KARP was predominantly present in the with distinct at the plasma membrane as in A. we membrane of E3KARP in the of (Fig. A and and However, this requires that of E3KARP. at the region was not for E3KARP. showed that NHE3 and E3KARP not the cells, but co-localized at the plasma membrane as as within the but not in the juxtanuclear with the of and E3KARP not with the E3KARP to in to cAMP. by et al. D. M. A. J. Biol. 1997; PubMed Scopus Google Scholar) and al. A. C. E. D. C. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that the human homolog of NHERF, binds to the Since both E3KARP and NHERF interact with NHE3 and have the function in the cAMP-dependent inhibition of NHE3, we ezrin also interact with E3KARP. The was with by in and was used as a for NHERF (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar, 6Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar) expressed as a fusion protein was used as a for the A that the to but not interaction between the of ezrin and E3KARP. The interaction of the with E3KARP was as as that with determine which of E3KARP binds to the interaction between the and the of E3KARP expressed as fusion proteins was by As shown in the not bind to the PDZ domains (P1 and P2) or to as one However, the to the C-terminal and domains of E3KARP. the not interact with the which is the carboxyl-terminal aa The of binding to the construct that the is within the carboxyl-terminal aa of E3KARP (Fig. the distribution of E3KARP and ezrin in cells. both proteins were expressed in the as membrane was also of ezrin and E3KARP that ezrin and E3KARP are co-localized in the plasma with for not the distribution of E3KARP or ezrin or the of these two In this study, we the interaction between NHE3 and E3KARP. We to the PDZ domains in E3KARP to the interaction with NHE3. This was not PDZ domains were shown to be the for but also one of the clones obtained in two-hybrid the second PDZ domain and a of the of E3KARP (5Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.-M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). on we that NHE3 interact with E3KARP the second PDZ on interaction PDZ domains showed that distinct PDZ domains interact with with a sequence of E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (897) Google Scholar, 12Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1628) Google Scholar). However, PDZ domains bind other PDZ the PDZ domain of bind to the second PDZ domain of in Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of E3KARP with NHE3 represents a new mode of binding for a PDZ In this study, we found that the second PDZ domain was a major in the interaction with the NHE3 but the second PDZ domain was not for the interaction. the domain and the of E3KARP a unit that with NHE3. This is not the first a PDZ domain does not in In other PDZ domains were required for interaction R.A. E. D. J. Biol. 1996; PubMed Scopus Google Scholar, P. G. A. G. J. G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). binds to the first and second PDZ domains of a human homolog of as a but not to PDZ domains R.A. E. D. J. Biol. 1996; PubMed Scopus Google Scholar). binding of a major to the PDZ protein requires two PDZ domains of P. G. A. G. J. G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). However, in this study, the domain and also the domain of E3KARP binding that were not and we this as a of the in The interaction of the NHE3 with E3KARP from previously in that it required the second PDZ domain and a non-PDZ the C-terminal the second PDZ domain of E3KARP does not in is not clear. The domain of E3KARP is shown to interact with an internal domain of NHE3 between aa 585 and 660 of the NHE3 C-terminal Since the E3KARP binding to was that to it is that the domain between aa 660 and 711 may also in binding to E3KARP. the of the domain within the NHE3 was limited to the we were not to the rabbit NHE3 C-terminal constructs as recombinant We to as and fusion proteins in E. coli, but these were in either the of or of of the and amino acid of NHE3 that are in the interaction with E3KARP requires it is that of the domain between aa and the cAMP-dependent inhibition of NHE3 (13Cabado A.G., Yu, F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Kurashima et al. (4Kurashima K., Yu, F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) identified of NHE3 as the of phosphorylation in NHE3 by PKA. However, of not the cAMP-dependent inhibition of NHE3 but the of the inhibition by In to the inhibition of NHE3 by of and were is not phosphorylated by PKA. The role of but it is to that may in the interaction with the regulatory that NHERF also binds to the region in NHE3 as E3KARP. and C. this is not the in which a PDZ domain with an internal the second PDZ domain of the protein binds an internal the of the 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). PDZ proteins function as scaffold proteins, in of proteins that interact with PDZ and in at sites interactions with the PDZ domains in the protein E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (897) Google H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1628) Google Scholar). of PS120 fibroblasts that E3KARP is in the (Fig. In addition, was distinct membrane with E3KARP. How E3KARP the cAMP effect on NHE3 is not However, it may of E3KARP in to phosphorylation by PKA. However, we found in this that distribution of E3KARP was not in the presence of We also that NHERF which is to E3KARP was not affected by The of of E3KARP is consistent with the that the phosphorylation level of protein was in to E. J. and C. in This showed that and E3KARP are co-localized at the plasma membrane, consistent with the interaction between and and the of NHE3 by cAMP. However, the distribution of in PS120 fibroblasts is distinct from that of with at and on the The is to NHE3 proteins present in S. Yu, K. Lukacs G. K. Orlowski J. Grinstein S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of was not by the presence of or of E3KARP. These show that the inhibition of NHE3 in to an elevated cAMP level may not of either NHE3 or E3KARP within the of However, we a small in E3KARP or NHE3 distribution by and this requires other Since PDZ proteins function as scaffold proteins, we have been a model in which E3KARP NHERF as a We E3KARP and NHERF Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), but found that these regulatory proteins are not G. A. Weinman E.J. C.H.C. 1998; Full Text PDF Google Scholar). was shown to bind to ezrin and other proteins D. M. A. J. Biol. 1997; PubMed Scopus Google Scholar, A. C. E. D. C. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Since ezrin has been shown to directly bind PKA J. M. C. P.H. J. 1997; PubMed Scopus Google Scholar), the binding of NHERF to ezrin suggested that the may be a between NHE3 and PKA D. M. A. J. Biol. 1997; PubMed Scopus Google Scholar, A. C. E. D. C. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). E3KARP and NHERF share a homology, and we E3KARP is an showed that the N-terminal 302 aa of human ezrin bind to both NHERF and E3KARP with We found that the interaction with ezrin the C-terminal of E3KARP. the to the C-terminal aa (Fig. a showed that ezrin also binds to the carboxyl-terminal aa of human D. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). A for proteins with a not proteins other NHERF and that this domain of E3KARP may a The of the PKA-dependent inhibition of NHE3 is to be The of that the increase in phosphorylation of NHE3 is essential for this inhibition (4Kurashima K., Yu, F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). the presence of a regulatory protein, either E3KARP or NHERF, is also Since ezrin is an that binds to the regulatory of PKA J. M. C. P.H. J. 1997; PubMed Scopus Google Scholar), the binding of ezrin to NHERF suggested a model in which NHERF may scaffold NHE3 and ezrin, which in PKA into the vicinity of NHE3, PKA to NHE3. the phosphorylation of NHE3 by PKA and the presence of the regulatory proteins are We are to Donowitz for and to for the human ezrin
Yun et al. (Thu,) studied this question.