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• W2065839189 abstract "Merlin, the neurofibromatosis 2 tumor suppressor protein, has two major isoforms with alternate C termini and is related to the ERM (ezrin, radixin, moesin) proteins. Regulation of the ERMs involves intramolecular and/or intermolecular head-to-tail associations between family members. We have determined whether merlin undergoes similar interactions, and our findings indicate that the C terminus of merlin isoform 1 is able to associate with its N-terminal domain in a head-to-tail fashion. However, the C terminus of isoform 2 lacks this property. Similarly, the N terminus of merlin can also associate with C terminus of moesin. We have also explored the effect of merlin self-association on binding to the regulatory cofactor of Na+-H+exchanger (NHE-RF), an interacting protein for merlin and the ERMs. Merlin isoform 2 captures more NHE-RF than merlin isoform 1 in affinity binding assays, suggesting that in full-length merlin isoform 1, the NHE-RF binding site is masked because of the self-interactions of merlin. Treatment with a phospholipid known to decrease self-association of ERMs enhances the binding of merlin isoform 1 to NHE-RF. Thus, although isoform 1 resembles the ERM proteins, which transition between inactive (closed) and active (open) states, isoform 2 is distinct, existing only in the active (open) state and presumably constitutively more available for interaction with other protein partners. Merlin, the neurofibromatosis 2 tumor suppressor protein, has two major isoforms with alternate C termini and is related to the ERM (ezrin, radixin, moesin) proteins. Regulation of the ERMs involves intramolecular and/or intermolecular head-to-tail associations between family members. We have determined whether merlin undergoes similar interactions, and our findings indicate that the C terminus of merlin isoform 1 is able to associate with its N-terminal domain in a head-to-tail fashion. However, the C terminus of isoform 2 lacks this property. Similarly, the N terminus of merlin can also associate with C terminus of moesin. We have also explored the effect of merlin self-association on binding to the regulatory cofactor of Na+-H+exchanger (NHE-RF), an interacting protein for merlin and the ERMs. Merlin isoform 2 captures more NHE-RF than merlin isoform 1 in affinity binding assays, suggesting that in full-length merlin isoform 1, the NHE-RF binding site is masked because of the self-interactions of merlin. Treatment with a phospholipid known to decrease self-association of ERMs enhances the binding of merlin isoform 1 to NHE-RF. Thus, although isoform 1 resembles the ERM proteins, which transition between inactive (closed) and active (open) states, isoform 2 is distinct, existing only in the active (open) state and presumably constitutively more available for interaction with other protein partners. neurofibromatosis 2 regulatory cofactor of Na+-H+ exchanger affinity co-electrophoresis phosphatidylinositol 4,5-bisphosphate glutathione S-transferase amino acid(s) glutathione polyacrylamide gel electrophoresis Merlin is the tumor suppressor protein deficient in neurofibromatosis 2 (NF2),1 a dominantly inherited disorder characterized by bilateral vestibular schwannomas and other brain tumors (1Martuza R.L. Eldridge R. N. Engl. J. Med. 1988; 318: 684-688Crossref PubMed Scopus (387) Google Scholar, 2Kaiser-Kupfer M.I. Freidlin V. Datiles M.B. Edwards P.A. Sherman J.L. Parry D. McCain L.M. Eldridge R. Arch. Ophthalmol. 1989; 107: 541-544Crossref PubMed Scopus (90) Google Scholar). Merlin has a striking similarity in sequence and structure with ezrin, radixin, and moesin, commonly referred to as the ERM proteins. The ERM proteins share ∼78% amino acid identity with each other, and all three are 45–47% identical to merlin (3Trofatter J.A. MacCollin M.M. Rutter J.L. Murrell J.R. Duyao M.P. Parry D.M. Eldridge R. Kley N. Menon A.G. Pulaski K. Haase V.H. Ambrose C.M. Munroe D. Bove C. Haines J.L. Martuza R.L. MacDonald M.E. Seizinger B.R. Short M.P. Buckler A.J. Gusella J.F. Cell. 1993; 72: 791-800Abstract Full Text PDF PubMed Scopus (1080) Google Scholar). Like the ERM proteins and protein 4.1, merlin possesses a FERM (protein 4.1, ezrin, radixin,moesin) domain (∼270 amino acids defining membership in the protein 4.1 superfamily) in its N-terminal half, followed by a long α-helical segment and a charged C-terminal domain (4Chishti A.H. Kim A.C. Marfatia S.M. et al.Trends Biochem. Sci. 1998; 23: 281-282Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). TheNF2 gene comprises 17 exons with alternative splicing of the penultimate exon producing two major merlin isoforms. Isoform 1 is a 595-amino acid protein produced from exons 1–15 and exon 17. Isoform 2 results from the presence of the alternatively spliced exon 16, which alters the C terminus of the protein to produce a 590-amino acid protein identical to isoform 1 over the first 579 residues (5Haase V.H. Trofatter J.A. McCollin M. Tarttelin E. Gusella J.F. Ramesh V. Hum. Mol. Genet. 1994; 3: 407-411Crossref PubMed Scopus (38) Google Scholar, 6Arakawa H. Hayashi N. Nagase H. Ogawa M. Nakamura Y. Hum. Mol. Genet. 1994; 3: 565-568Crossref PubMed Scopus (90) Google Scholar, 7Pykett M.J. Murphy M. Harnish P.R. George D.L. Hum. Mol. Genet. 1994; 3: 559-564Crossref PubMed Scopus (69) Google Scholar). Mutational analysis has revealed a wide variety of mutations in the germline and tumors of NF2 patients as well as in sporadic schwannomas and meningiomas, confirming merlin's tumor suppressor function (8Gusella J.F. Ramesh V. McCollin M. Jacoby L.B. Curr. Opin. Genet. Dev. 1996; 6: 87-92Crossref PubMed Scopus (48) Google Scholar). ERM proteins act as linkers between integral membrane proteins and the actin cytoskeleton (9Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. Tsukita S. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (676) Google Scholar). Proteins identified as ligands for ERM proteins include CD44, CD43, ICAM1, ICAM2, and actin (9Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. Tsukita S. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (676) Google Scholar, 10Turunen O. Wahlstršm T. Vaheri A. J. Cell Biol. 1994; 126: 1445-1453Crossref PubMed Scopus (343) Google Scholar, 11Pestonjamasp K. Amieva M.R. Strassel C.P. Nauseef W.M. Furthmayr H. Luna E.J. Mol. Biol. Cell. 1995; 6: 247-259Crossref PubMed Scopus (154) Google Scholar, 12Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita S. J. Cell Biol. 1998; 140: 885-895Crossref PubMed Scopus (503) Google Scholar, 13Heiska L. Alfthan K. Gronholm M. Vilja P. Vaheri A. Carpen O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). We and others have recently identified the human homologue of a regulatory cofactor for Na+-H+ exchanger (NHE-RF) as a novel interactor for the conserved N terminus of merlin and ERM proteins (14Murthy A. Gonzalez-Agosti C. Cordero E. Pinney D. Candia C. Solomon F. Gusella J. Ramesh V. J. Biol. Chem. 1998; 273: 1273-1276Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 15Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (512) Google Scholar). In addition to interacting with many binding partners, the ERM proteins are capable of forming homo- and heterotypic associations between family members (16Gary R. Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10846-10850Crossref PubMed Scopus (93) Google Scholar, 17Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (177) Google Scholar). Indeed, several recent studies performed on the regulation of ERM proteins suggest that the availability of ERM domains to binding partners is controlled by self-association of the N-terminal and C-terminal regions (13Heiska L. Alfthan K. Gronholm M. Vilja P. Vaheri A. Carpen O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 18Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 19Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar). Thus the ERM proteins can exist in the “closed” state, where the N- and C-terminal regions undergo an intramolecular interaction, masking the respective ligand-binding site. This closed state can be converted to the “open” state in which intramolecular interaction is disrupted by a variety of cellular signals, including Rho-mediated signaling and the phospholipid PIP2. In vitro binding studies performed with merlin isoforms suggested that the C terminus of isoform 1 can interact with its N terminus, and the C terminus of isoform 2 lacked this property (20Sherman L. Xu H.M. Geist R.T. Saporito-Irwin S. Howells N. Ponta H. Herrlich P. Gutmann D.H. Oncogene. 1997; 15: 2505-2509Crossref PubMed Scopus (201) Google Scholar). Homotypic interaction of merlin isoform 1 and heterotypic interaction between merlin and the ERM proteins have also been reported recently by yeast two hybrid and blot overlay assays (21Gronholm M. Sainio M. Zhao F. Heiska L. Vaheri A. Carpen O. J. Cell Sci. 1999; 112: 895-904PubMed Google Scholar, 22Huang L. Ichimaru E. Pestonjamasp K. Cui X. Nakamura H. Lo G.Y.H. Lin F.I.K. Luna E.J. Furthmayr H. Biochem. Biophys. Res. Commun. 1998; 248: 548-553Crossref PubMed Scopus (37) Google Scholar). In view of the importance of self-association in regulation of the ERM proteins, we have used affinity co-electrophoresis (ACE) assays to explore the capacity of the two major merlin isoforms to self-associate and to interact with a representative ERM protein. The uniqueness of this assay is the ability to determine the dissociation constants of the observed interactions. Although, like the ERMs, the C terminus of merlin isoform 1 interacts in a head-to-tail fashion with its N-terminal domain, the C terminus of isoform 2 lacks this property. The N terminus of merlin is also able to associate heterotypically with the C terminus of moesin. Furthermore, in affinity binding experiments we observe that full-length merlin isoform 2 is able to capture greater quantities of NHE-RF than full-length merlin isoform 1, consistent with the notion that the ligand binding is suppressed by the self-interaction of merlin isoform 1. In addition, the interaction between merlin isoform 1 and NHE-RF is enhanced in the presence of the phosphoinositide PIP2. Thus, merlin isoform 1 behaves like the ERM proteins in its interdomain interaction and its regulation by phospholipids, but merlin isoform 2 does not exhibit this property and is always available for interaction with other ligands. The different regulation of inter- and intramolecular domain interactions of the isoforms of merlin could play an essential role in its tumor suppressor function. Full-length isoform 1 (aa 1–595), full-length isoform 2 (aa 1–590), N-terminal (aa 1–332), and C-terminal (isoform 1 aa 340–595; isoform 2 aa 340–590; common to both isoforms aa 340–579) portions of merlin were expressed as glutathione S-transferase (GST) fusion proteins in pGEX2T. Also, full-length moesin (aa 1–577), N-terminal (aa 1–332), and C-terminal (aa 307–577) portions of moesin were expressed as GST fusion proteins in pGEX4T1. Similarly, full-length NHE-RF (aa 1–338) was expressed as a GST fusion protein. Expression and purification of the GST fusion proteins were performed as described previously for merlin (23Gonzalez-Agosti C. Xu L. Pinney D. Beauchamp R. Hobbs W. Gusella J. Ramesh V. Oncogene. 1996; 13: 1239-1247PubMed Google Scholar) and using standard methods for moesin and NHE-RF. In addition, full-length merlin isoform 1 and 2 were cloned into the mammalian expression vector pcDNA 3 engineered to have a FLAG tag at the N terminus. These constructs were transiently expressed in Cos-7 cells as described previously (24Xu L. Gonzalez-Agosti C. Beauchamp R. Pinney D. Sterner C. Ramesh V. Exp. Cell. Res. 1998; 238: 231-240Crossref PubMed Scopus (30) Google Scholar). The polyclonal anti-merlin antibody (N21) and the polyclonal anti-GST antibody have been described previously (23Gonzalez-Agosti C. Xu L. Pinney D. Beauchamp R. Hobbs W. Gusella J. Ramesh V. Oncogene. 1996; 13: 1239-1247PubMed Google Scholar,25Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). A rabbit polyclonal antibody IC270 was raised against the GST-NHE-RF fusion protein (aa 270–358). The anti-FLAG antibody M2 was commercially obtained (Kodak, IBI). For affinity co-electrophoresis (ACE), purified GST fusion protein products of merlin and moesin were thrombin cleaved. ACE gels were prepared using 1% low melting point agarose in 125 mm potassium acetate, 50 mmHepes, pH 7.5, and carried out as described (25Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 26Herndon M.E. Lander A.D. Jackson P.I. Gallagher J.T. A Laboratory Guide to Glycoconjugate Analysis. Birkhauser Verlag AG, Basel, Switzerland1997: 379-398Crossref Google Scholar). Gels were run at 60 volts for 4 h, and the proteins were then transferred to nitrocellulose by capillary action and analyzed by immunoblotting using an affinity eluted N terminus-specific anti-merlin antibody (N21) at a dilution of 1:100 and protein A conjugated to 125I. Retardation coefficients were calculated as described previously (27Lim W.A. Sauer R.T. Lander A.D. Methods Enzymol. 1991; 208: 196-212Crossref PubMed Scopus (58) Google Scholar). Dissociation constants were calculated from nonlinear, least squares fitting of plots of corrected retardation coefficient versusconcentration of retarding protein (27Lim W.A. Sauer R.T. Lander A.D. Methods Enzymol. 1991; 208: 196-212Crossref PubMed Scopus (58) Google Scholar, 28SanAntonio J.D. Slover J. Lawler J. Karnovsky M.J. Lander A.D. Biochemistry. 1993; 32: 4746-4755Crossref PubMed Scopus (81) Google Scholar). Data were then fit, using a nonlinear least squares approach (Kaleidagraph, Synergy Software), to the equation r = R ∞/[1 + (K d/[P tot])], wherer = retardation coefficient and [P tot] = protein concentration in a given lane of an ACE gel. The variables that were fit simultaneously wereK d, the dissociation constant, andR ∞, the maximum value of R. Data from two independent experiments were used for calculating dissociation constants, and the K d value is shown with S.E. ZR-75-B cells were lysed in Brij lysis buffer (50 mm Tris, 150 mm NaCl, 1 mm EDTA, pH 8.0, 30% glycerol, 1% Brij 96) containing a 1× protease inhibitor mixture (Roche Molecular Biochemicals), and the lysate was incubated with 600 pmol of GST-merlin immobilized on glutathione (GSH)-Sepharose 4B beads. The beads were washed extensively with phosphate-buffered saline containing Pefabloc, resuspended in Laemmli loading buffer, subjected to 10% SDS-PAGE, and immunoblotted with IC270 antiserum (1:1000). In some experiments, GST-merlin immobilized on beads was incubated with ZR-75-B lysate along with 50 μl/ml phosphatidyl serine or PIP2 (Sigma). Each phospholipid was dissolved in distilled water to a final concentration of 1 mg/ml and sonicated three times each for 10 s. Cos-7 cells expressing merlin isoforms 1 and 2 as FLAG-tagged proteins were lysed in Nonidet P-40 lysis buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 0.5% Nonidet P-40) containing a 1× protease inhibitor mixture as described above. The lysates were incubated with 600 pmol of GST-NHE-RF full-length fusion protein or GST protein alone coupled to GSH-Sepharose 4B beads. The beads were washed as described above, and the separated proteins were immunoblotted with the anti-FLAG antibody M2. To determine whether merlin as an ERM family member is capable of self-association and to ascertain whether the two major isoforms of merlin differ from each other in this property, we used the technique of ACE. Using this technique we were able to demonstrate direct binding between the N- and C termini of merlin isoforms in solution and to measure the strength of the binding. Briefly, the thrombin cleaved N- (aa 1–332) and C-terminal (iso 1, aa 340–595 or iso 2, aa 340–590) polypeptides were subjected to affinity electrophoresis in 1% agarose gel in physiological buffer. The N terminus of merlin (at 125 nm) was loaded into a long transverse slot. The C terminus of either isoform 1 or 2 of merlin was cast (in agarose) into nine rectangular wells at concentrations ranging from 750 to 0 nm. The anode was placed so that the more rapidly migrating N-domain passes through the zones containing the C-domain during most of the electrophoresis run. The mobility of the N-domain was then detected by transferring the proteins to nitrocellulose and probing with an N-terminal-specific antibody. Fig. 1 A demonstrates the interaction of the N terminus of merlin with the C terminus of merlin isoform 1, where migrating N-domain encountered the C-domain. The migration of the former was retarded in a manner that varied directly with the concentration of the latter. In contrast, the migration of the N-domain of merlin was not retarded by the C-domain of merlin isoform 2 over the same range of concentrations (Fig.1 B). Because we were able to demonstrate a difference in self-association between the two isoforms of merlin that differ from each other only at the extreme C terminus, we further sought to determine whether the C-domain of merlin lacking both exons 16 and 17 is capable of self-interaction. To address this question we performed the same type of experiment described above except the C-domain of merlin spanned the common region aa 340–579. The results shown in Fig. 1 Cindicate that the migration of the N terminus was not retarded by this C-domain. These data suggest that the domain responsible for the self-association of the N terminus of merlin to the C terminus of merlin isoform 1 includes, at least in part, the last 16 amino acids (aa 580–595) specific to this isoform. To evaluate whether merlin can interact in a heterotypic fashion with other ERM family members, we performed ACE experiments where the N-domain of merlin was used as the faster migrating protein passing through the zones containing the C-domain of moesin (aa 305–557). Fig.1 D shows the interaction of merlin and moesin by the retardation of migration of the N terminus of merlin. The migration of purified GST used as a control protein was not affected by the merlin C-domain isoform 1 and moesin C-domain over the same range of concentrations (Fig. 1, E and F). From measurements of mobility retardation in Fig. 1 (A–F), we can calculate the dissociation constant for the interaction of the N- and C-terminal polypeptides of merlin isoforms 1 and 2 (homotypic) and for the interaction of merlin to moesin (heterotypic). To avoid problems arising from the saturation of the films, ImageQuant software (Molecular Dynamics) was used to determine the true midpoint of each of the bands (28SanAntonio J.D. Slover J. Lawler J. Karnovsky M.J. Lander A.D. Biochemistry. 1993; 32: 4746-4755Crossref PubMed Scopus (81) Google Scholar). Fig. 2 shows the analyses of the six representative experiments. For every gel, retardation coefficients (R) were determined for each C-terminal protein concentration tested. Dissociation constants were measurable for N-merlin interaction with the C-merlin isoform 1 and N-merlin interaction with the C-moesin by fitting the data to the equationr = R ∞/[1 =(K d,app/[proteintot])] (see “Experimental Procedures”). Merlin isoform 1 can self-interact with a K d of 49.0 ± 6.45 nm, whereas merlin interacts with moesin with a K d of 28.5 ± 6.5 nm. By contrast, no binding was measurable with merlin isoform 2 and control proteins. We recently identified NHE-RF, a regulatory factor for the Na+-H+ exchanger isoform 3 (NHE3), as an interacting protein for merlin in a two-hybrid screen and demonstrated that NHE-RF can bind to merlin, moesin, and radixin via their conserved N-terminal regions (14Murthy A. Gonzalez-Agosti C. Cordero E. Pinney D. Candia C. Solomon F. Gusella J. Ramesh V. J. Biol. Chem. 1998; 273: 1273-1276Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). It is well documented that the binding of ERM proteins to their ligands is suppressed in the native full-length protein (13Heiska L. Alfthan K. Gronholm M. Vilja P. Vaheri A. Carpen O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 18Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar, 19Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar), a phenomenon explained by the interdomain interactions of the ERM proteins that could compete with the ligand binding (29Reczek D. Bretscher A. J. Biol. Chem. 1998; 273: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). We therefore investigated whether NHE-RF displays differential binding with isoforms 1 and 2 of merlin by performing affinity precipitation experiments. Briefly, equal quantities of merlin full-length isoforms 1 and 2, N-domain, C-domain of both isoforms 1 and 2, expressed as GST fusion proteins, were bound to Sepharose beads and incubated with equal amount of ZR-75-B cell lysates. After extensive washes, the coupled proteins were removed from the beads by boiling and were detected on Western blots using a specific polyclonal antibody against NHE-RF. The results shown in Fig.3 demonstrate that the N-domain and full-length isoform 2 of merlin exhibit a greater affinity for NHE-RF than merlin isoform 1. As expected NHE-RF did not bind to either the C-domain of both isoforms of merlin or the GST control protein. These results were further confirmed by at least three independent experiments. To confirm the differential binding of merlin isoforms to NHE-RF, affinity binding assays were performed utilizing merlin isoforms expressed as FLAG-tagged proteins in Cos-7 cells and GST fusion protein of NHE-RF. The expression of FLAG-tagged merlin isoforms in Cos-7 cells were examined with an anti-FLAG antibody (M2) and found to be equally expressed (Fig. 4, lanes 1 and2). Cos-7 cell lysates expressing approximately the same amount of the isoforms were incubated with 600 pmol of GST-NHE-RF beads. The bound proteins were separated on a 7.5% SDS-PAGE and probed with M2 antibody (Fig. 4). Merlin isoforms expressed in mammalian cells also revealed a differential binding to NHE-RF, and the analysis of the supernatants that were not bound to the beads clearly showed that NHE-RF beads capture 3–5-fold more merlin isoform 2 than isoform 1 in duplicate set of experiments. These data are consistent with the ACE results supporting that merlin isoform 2 exists constitutively in an open conformation that allows its ligand, NHE-RF, to interact without being hindered by the interdomain interaction that occurs in merlin isoform 1. Phosphatidylinositol 4-phosphate and PIP2 enhance the interaction of ERM proteins to its ligands CD44, ICAM-1, and ICAM-2 (13Heiska L. Alfthan K. Gronholm M. Vilja P. Vaheri A. Carpen O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 18Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar). ERM proteins bind to phosphatidylinositol 4-phosphate and PIP2 (30Niggli V. Andreoli C. Roy C. Manget P. FEBS Lett. 1995; 376: 172-176Crossref PubMed Scopus (162) Google Scholar), and it is believed that this binding evokes conformational changes in these proteins in a manner similar to the regulation of vinculin activity by PIP2 (31Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (453) Google Scholar). We therefore examined whether PIP2 influences the binding of NHE-RF to merlin isoforms. For this, PIP2 or phosphatidyl serine was included in the affinity precipitation assays. In the presence of PIP2 the amount of NHE-RF bound to merlin isoform 1 significantly increased (Fig. 5). The intensity of the bands was further analyzed by densitometric scanning of the autorads using transmittance analysis (Fluor-S, Multiimager, Bio-Rad). Results from three independent experiments revealed a 3-fold increase (3.10 ± 1.04) in binding of NHE-RF to merlin isoform 1 in the presence of PIP2. The binding of NHE-RF to either the N-domain of merlin or merlin isoform 2 (1.07 ± 0.45) was not influenced by PIP2 (Fig. 5). The control phospholipid phosphatidyl serine did not enhance the NHE-RF binding to merlin isoform 1 (Fig. 5). Despite an overall structural similarity to the ERM proteins, merlin differs from these relatives in having two isoforms with alternative C termini and in having a demonstrated tumor suppressor function. Both isoforms are expressed at the RNA and protein level in a variety of cell lines examined including NF2 target cells such as Schwann and meningeal cells (Refs. 6Arakawa H. Hayashi N. Nagase H. Ogawa M. Nakamura Y. Hum. Mol. Genet. 1994; 3: 565-568Crossref PubMed Scopus (90) Google Scholar and 7Pykett M.J. Murphy M. Harnish P.R. George D.L. Hum. Mol. Genet. 1994; 3: 559-564Crossref PubMed Scopus (69) Google Scholar and our unpublished data). 2Solomon, F., personal communication. Work from other laboratories has demonstrated the interdomain interaction of merlin using yeast two hybrid, blot overlay, co-immunoprecipitation, andin vitro binding assays (20Sherman L. Xu H.M. Geist R.T. Saporito-Irwin S. Howells N. Ponta H. Herrlich P. Gutmann D.H. Oncogene. 1997; 15: 2505-2509Crossref PubMed Scopus (201) Google Scholar, 21Gronholm M. Sainio M. Zhao F. Heiska L. Vaheri A. Carpen O. J. Cell Sci. 1999; 112: 895-904PubMed Google Scholar, 22Huang L. Ichimaru E. Pestonjamasp K. Cui X. Nakamura H. Lo G.Y.H. Lin F.I.K. Luna E.J. Furthmayr H. Biochem. Biophys. Res. Commun. 1998; 248: 548-553Crossref PubMed Scopus (37) Google Scholar). Employing the technique of ACE, we not only demonstrate the difference between merlin isoforms in their interdomain interaction but also define the affinities of these interactions. The affinities that we have observed for merlin self-interaction and for the interaction of merlin with moesin are quite comparable with that reported for radixin using similar analysis (25Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Because the C-terminal construct of merlin containing the common region (aa 340–579) did not exhibit the self-interaction, we believe that the isoform 1-specific C-terminal residues (aa 580–595) are critical for the interdomain binding. This is in agreement with a previous report suggesting that extreme C-terminal protein sequences encoded by exon 17 is critical for the interdomain interaction (20Sherman L. Xu H.M. Geist R.T. Saporito-Irwin S. Howells N. Ponta H. Herrlich P. Gutmann D.H. Oncogene. 1997; 15: 2505-2509Crossref PubMed Scopus (201) Google Scholar). It is well established that the interaction of ERM proteins with their membrane partners, as well as with the actin cytoskeleton, is suppressed in the full-length molecule, a phenomenon explained by intramolecular self-association that masks the binding sites for other ligands (32Tsukita S. Yonemura S. Tsukita S. Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 33Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (329) Google Scholar). In the presence of phospholipids such as phosphatidylinositol 4-phosphate or PIP2, the interdomain interaction is disrupted, thus exposing the ligand binding sites (13Heiska L. Alfthan K. Gronholm M. Vilja P. Vaheri A. Carpen O. J. Biol. Chem. 1998; 273: 21893-21900Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar,18Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar). Interdomain interaction also blocks the binding of full-length ezrin and radixin to NHE-RF compared with their N termini (29Reczek D. Bretscher A. J. Biol. Chem. 1998; 273: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar).2 In our earlier studies (14Murthy A. Gonzalez-Agosti C. Cordero E. Pinney D. Candia C. Solomon F. Gusella J. Ramesh V. J. Biol. Chem. 1998; 273: 1273-1276Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), we failed to note a similar difference in binding to NHE-RF between the full-length merlin isoform 1 and the N-domain of merlin (aa 1–332). This apparent discrepancy could be explained by the fact that the previous studies were designed to answer qualitatively whether merlin binds to NHE-RF. However, the present studies were done in a quantitative fashion to address the differences in binding between the two isoforms of merlin. Our results demonstrate that the binding of merlin isoform 1 to its ligand NHE-RF is suppressed in the full-length molecule, and in the presence of PIP2 this suppression is relieved. Merlin isoforms expressed in mammalian cells show the same difference in their binding to NHE-RF as the bacterially expressed proteins. The fact that merlin isoforms expressed in mammalian cells show differential binding to NHE-RF suggests that this may have functional significance in vivo. The studies performed here illustrate the differences in the ability of the two alternatively spliced isoforms of merlin to interact with NHE-RF and further show that a phospholipid such as PIP2can regulate the interaction of merlin isoform 1 to NHE-RF. Thus these results document that merlin isoform 1 behaves in a manner similar to its ERM relatives, whereas merlin isoform 2 behaves distinctly and binds to NHE-RF more efficiently. Similarly, betaII-spectrin, a C-terminal interactor of merlin, has been shown to interact to a greater extent with the C terminus of merlin isoform 2 than with the C terminus of isoform 1 (34Scoles D.R. Huynh D.P. Morcos P.A. Coursell E.R. Robinson N.G.G. Tamanoi F. Pulst S.M. Nat. Genet. 1998; 18: 354-359Crossref PubMed Scopus (128) Google Scholar). Phosphorylation of a critical Thr residue at the C terminus of the ERM proteins has been implicated in stabilizing the open conformation of these proteins (19Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (721) Google Scholar). This Thr residue is conserved in both isoforms of merlin; however, further studies are required to understand whether the phosphorylation of this residue is involved in regulating the intramolecular interaction of merlin isoform 1 and what role it might play in the function of merlin isoform 2. Both NHE-RF and the related NHE-RF2 possess two PDZ domains known to mediate protein-protein interactions. The interaction of NHE-RF and NHE-RF2 with merlin and the ERMs is not mediated by the PDZ domains (14Murthy A. Gonzalez-Agosti C. Cordero E. Pinney D. Candia C. Solomon F. Gusella J. Ramesh V. J. Biol. Chem. 1998; 273: 1273-1276Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 29Reczek D. Bretscher A. J. Biol. Chem. 1998; 273: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 35Yun C.H. Lamprecht G. Foster D.V. Sidor A. J. Biol. Chem. 1998; 273: 25856-25863Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). However, the PDZ domains of both NHE-RF and NHE-RF2 can interact with several other membrane proteins, such as Na+-H+ exchanger isoform 3, the β2-adrenergic receptor, the purinergic P2Y1 receptor, and the cystic fibrosis transmembrane conductance regulator, which functions as a Cl− channel (35Yun C.H. Lamprecht G. Foster D.V. Sidor A. J. Biol. Chem. 1998; 273: 25856-25863Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 36Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (371) Google Scholar). Thus, NHE-RF and NHE-RF2 appear to act as multifunctional adaptor proteins that may link merlin and the ERM proteins to different ion channels and receptors, providing many new possibilities for effects on intracellular signaling. Because merlin isoform 2 exists always in the open state, its interaction with NHE-RF and potentially with other merlin interactors may in fact occur when the equivalent sites in merlin isoform 1 and in the ERM proteins are masked in the closed state. The strategy of comparing merlin with the related ERM proteins can be expected to produce similarities that are instructive concerning the overall function of these types of proteins and differences that could reveal the special tumor suppressor activity of merlin. In this study, we have observed both. The behavior of merlin isoform 1 with respect to interdomain interactions suggests that its regulation is similar to the regulation of ERM protein interactions. Moreover, the interaction of merlin with the ERM proteins suggests that these proteins could also be involved in mutual regulation of each other's activities. Although the full range of merlin interactions with other proteins remains to be delineated, it is likely that merlin sits within a web of interactions comprising multiple partners and signaling pathways, some of which are shared with the ERM family members. Interestingly, studies of theDrosophila homologue of merlin suggest that its growth suppression properties reside within the conserved N-terminal domain of the protein (37Lajeunesse D.R. McCartney B.M. Fehon R.G. J. Cell Biol. 1998; 141: 1589-1599Crossref PubMed Scopus (119) Google Scholar). Thus, the distinct tumor suppressor role of merlin could lie either in the distinct regulation of isoform 2, which differs from that of isoform 1 and the ERM proteins, or in the participation of merlin but not the ERM proteins in a signaling pathway that is uniquely important to NF2 target cells such as Schwann and meningeal cells. We thank Dr. Frank Solomon for valuable discussions. We also thank Drs. Margaret Magendantz and Etchell Cordero for advice on the ACE experiments. The technical expertise of Denise Pinney and Cecilia Candia is gratefully acknowledged. We thank the members of our laboratory for helpful comments on the manuscript." @default.
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• W2065839189 title "Interdomain Interaction of Merlin Isoforms and Its Influence on Intermolecular Binding to NHE-RF" @default.
• W2065839189 cites W1967020063 @default.
• W2065839189 cites W1967941200 @default.
• W2065839189 cites W1971700401 @default.
• W2065839189 cites W1974264340 @default.
• W2065839189 cites W1975862854 @default.
• W2065839189 cites W1980364128 @default.
• W2065839189 cites W1986441957 @default.
• W2065839189 cites W1997224206 @default.
• W2065839189 cites W1998624086 @default.
• W2065839189 cites W2001449914 @default.
• W2065839189 cites W2024028178 @default.
• W2065839189 cites W2029863963 @default.
• W2065839189 cites W2036049934 @default.
• W2065839189 cites W2042202484 @default.
• W2065839189 cites W2051384784 @default.
• W2065839189 cites W2054223973 @default.
• W2065839189 cites W2065680809 @default.
• W2065839189 cites W2068473054 @default.
• W2065839189 cites W2080377199 @default.
• W2065839189 cites W2091962928 @default.
• W2065839189 cites W2092369394 @default.
• W2065839189 cites W2095359507 @default.
• W2065839189 cites W2112630320 @default.
• W2065839189 cites W2114964393 @default.
• W2065839189 cites W2117822148 @default.
• W2065839189 cites W2123117119 @default.
• W2065839189 cites W2141240174 @default.
• W2065839189 cites W2144476583 @default.
• W2065839189 cites W2154459309 @default.
• W2065839189 cites W2161807488 @default.
• W2065839189 cites W2167593330 @default.
• W2065839189 cites W2185731407 @default.
• W2065839189 cites W2213347258 @default.
• W2065839189 cites W2315552227 @default.
• W2065839189 cites W2415494887 @default.
• W2065839189 doi "https://doi.org/10.1074/jbc.274.48.34438" @default.
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