Dimerization of the Muscle-specific Kinase Induces Tyrosine Phosphorylation of Acetylcholine Receptors and Their Aggregation on the Surface of Myotubes

During development of the neuromuscular junction, neuronal splice variants of agrin initiate the aggregation of acetylcholine receptors on the myotube surface. The muscle-specific kinase is thought to be part of an agrin receptor complex, although the recombinant protein does not bind agrin with high affinity. To specify its function, we induced phosphorylation and activation of this kinase in the absence of agrin by incubating myotubes with antibodies directed against its N-terminal sequence. Antibody-induced dimerization of the muscle-specific kinase but not treatment with Fab fragments was sufficient to trigger two key events of early postsynaptic development: acetylcholine receptors accumulated into aggregates, and their β-subunits became phosphorylated on tyrosine residues. Heparin partially inhibited receptor aggregation induced by both agrin and anti-muscle-specific kinase antibodies. In contrast, it did not affect kinase or acetylcholine receptor phosphorylation. These data indicate that agrin induces postsynaptic differentiation by dimerizing the muscle-specific kinase. They also suggest that activation of the kinase domain can account for only part of agrin's effects. Dimerization of this molecule appears to activate an additional signal, most likely by organizing a scaffold for other postsynaptic proteins. During development of the neuromuscular junction, neuronal splice variants of agrin initiate the aggregation of acetylcholine receptors on the myotube surface. The muscle-specific kinase is thought to be part of an agrin receptor complex, although the recombinant protein does not bind agrin with high affinity. To specify its function, we induced phosphorylation and activation of this kinase in the absence of agrin by incubating myotubes with antibodies directed against its N-terminal sequence. Antibody-induced dimerization of the muscle-specific kinase but not treatment with Fab fragments was sufficient to trigger two key events of early postsynaptic development: acetylcholine receptors accumulated into aggregates, and their β-subunits became phosphorylated on tyrosine residues. Heparin partially inhibited receptor aggregation induced by both agrin and anti-muscle-specific kinase antibodies. In contrast, it did not affect kinase or acetylcholine receptor phosphorylation. These data indicate that agrin induces postsynaptic differentiation by dimerizing the muscle-specific kinase. They also suggest that activation of the kinase domain can account for only part of agrin's effects. Dimerization of this molecule appears to activate an additional signal, most likely by organizing a scaffold for other postsynaptic proteins. The basal membrane protein agrin plays a central role during the early phase of synaptic differentiation at the neuromuscular junction (1McMahan U.J. 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Neurosci. 1996; 15: 6767-6778Crossref Google Scholar) are able to induce the aggregation of AChRs 1The abbreviations used are: AChR, nicotinic acetylcholine receptor; DMEM, Dulbecco's modified Eagle's medium; MuSK, muscle-specific kinase; MASC, MuSK-accessory specificity component; mAb, monoclonal antibody; pAb, polyclonal antibody; RATL, rapsyn-associated transmembrane linker; s-agrin, soluble agrin; PAGE, polyacrylamide gel electrophoresis; TGFβR I, transforming growth factor β receptor I. 1The abbreviations used are: AChR, nicotinic acetylcholine receptor; DMEM, Dulbecco's modified Eagle's medium; MuSK, muscle-specific kinase; MASC, MuSK-accessory specificity component; mAb, monoclonal antibody; pAb, polyclonal antibody; RATL, rapsyn-associated transmembrane linker; s-agrin, soluble agrin; PAGE, polyacrylamide gel electrophoresis; TGFβR I, transforming growth factor β receptor I. and other synaptic proteins on the surface of myotubes (7Ferns M. Hoch W. Campanelli J.T. Rupp F. 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Rapsyn, a peripheral membrane protein closely associated with AChRs (12Froehner S.C. Gulbrandsen V. Hyman C. Jeng A.Y. Neubig R.R. Cohen J.B. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5230-5234Crossref PubMed Scopus (85) Google Scholar, 13Burden S.J. DePalma R.L. Gottesman G.S. Cell. 1983; 35: 687-692Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 14Froehner S.C. Annu. Rev. Neurosci. 1993; 16: 144-158Crossref Scopus (151) Google Scholar, 15Maimone M.M. Merlie J.P. Neuron. 1993; 11: 53-66Abstract Full Text PDF PubMed Scopus (89) Google Scholar), is an essential component of this pathway. In rapsyn-deficient mice, agrin is not able to induce the concentration of AChRs and other synaptic components (16Gautam M. Noakes P.G. Mudd J. Nichol M. Chu G.C. Sanes J.R. Merlie J.P. Nature. 1995; 377: 232-236Crossref PubMed Scopus (471) Google Scholar). Inhibitor studies suggest an important role of tyrosine phosphorylation in this pathway (17Wallace B.G. J. Cell Biol. 1994; 125: 661-668Crossref PubMed Scopus (99) Google Scholar). Agrin induces the tyrosine phosphorylation of the β-subunit of the AChR (18Wallace B.G. Qu Z. Huganir R.L. Neuron. 1991; 6: 869-878Abstract Full Text PDF PubMed Scopus (213) Google Scholar). It is unknown whether this modification is necessary for AChR aggregation. α-Dystroglycan, a component of the dystrophin-associated glycoprotein complex, has been identified as the most abundant agrin-binding protein on the myotube surface (19Hoch W. Campanelli J.T. Scheller R.H. J. Cell Biol. 1994; 126: 1-4Crossref PubMed Scopus (47) Google Scholar, 20Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar). However, the analysis of a series of agrin fragments has revealed no correlation between their binding to α-dystroglycan and their capability of inducing AChR aggregation (20Henry M.D. Campbell K.P. Curr. Opin. 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DiStefano P.S. Rojas E. Mattsson K. Compton D.L. Nunez L. Park J.S. Stark J.L. Gies D.R. Thomas S. Le Beau M.M. Fernald A.A. Copeland N.G. Jenkins N.A. Burden S.J. Glass D.J. Yancopoulos G.D. Neuron. 1995; 15: 573-584Abstract Full Text PDF PubMed Scopus (355) Google Scholar, 26Ganju P. Walls E. Brennan J. Reith A.D. Oncogene. 1995; 11: 281-290PubMed Google Scholar, 27Besser J. Zahalka M.A. Ullrich A. Mech. Dev. 1996; 59: 41-52Crossref PubMed Scopus (4) Google Scholar). In mice, deletion of this gene prevents the concentration of AChRs and other proteins at the contact site between motoneuron and muscle fiber and is therefore lethal (23DeChiara T.M. Bowen D.C. Valenzuela D.M. Simmons M.V. Poueymirou W.T. Thomas S. Kinetz E. Compton D.L. Rojas E. Park J.S. Smith C. DiStefano P.S. Glass D.J. Burden S.J. Yancopoulos G.D. Cell. 1996; 85: 501-512Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). MuSK is highly expressed in rat embryonic muscle and in the C2C12 mouse muscle cell line and colocalizes with AChRs at the neuromuscular junction (25Valenzuela D.M. Stitt T.N. DiStefano P.S. Rojas E. Mattsson K. Compton D.L. Nunez L. Park J.S. Stark J.L. Gies D.R. Thomas S. Le Beau M.M. Fernald A.A. Copeland N.G. Jenkins N.A. Burden S.J. Glass D.J. Yancopoulos G.D. Neuron. 1995; 15: 573-584Abstract Full Text PDF PubMed Scopus (355) Google Scholar, 27Besser J. Zahalka M.A. Ullrich A. Mech. Dev. 1996; 59: 41-52Crossref PubMed Scopus (4) Google Scholar). Several observations suggest an important role of MuSK in the agrin pathway (28Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar); incubation of myotubes with agrin causes the rapid tyrosine phosphorylation of MuSK (28Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). This reaction, a characteristic response of receptor tyrosine kinases to binding of their ligand (29Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4593) Google Scholar, 30Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1291) Google Scholar), is exclusively induced by biologically active fragments and isoforms of agrin. 2C. Hopf and W. Hoch, submitted for publication. 2C. Hopf and W. Hoch, submitted for publication. In addition, agrin can be cross-linked to MuSK expressed on myotubes (28Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). Upon transfection into the quail cell line QT-6, MuSK is concentrated in microaggregates together with rapsyn (31Gillespie S.K.H. Balasubramanian S. Fung E.T. Huganir R.L. Neuron. 1996; 16: 953-962Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Remarkably, the extracellular domain of the MuSK molecule is required for this interaction, which therefore must be indirect. It has been suggested that a hypothetical rapsyn-associated transmembrane linker (RATL) bridges these proteins (32Apel E.D. Glass D.J. Moscoso L.M. Yancopoulos G.D. Sanes J.R. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). Agrin does not bind to recombinant MuSK (28Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar) not a MuSK-accessory specificity component has been its activation by agrin (28Glass D.J. Bowen D.C. Stitt T.N. Radziejewski C. Bruno J. Ryan T.E. Gies D.R. Shah S. Mattsson K. Burden S.J. DiStefano P.S. Valenzuela D.M. DeChiara T.M. Yancopoulos G.D. Cell. 1996; 85: 513-523Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). To the role of MuSK in the agrin it is important to activate this molecule of agrin. In an a molecule of the extracellular domain of the receptor and the domain of MuSK has been expressed in The ligand to these myotubes induces the tyrosine phosphorylation of the receptor as as but not AChR aggregation D.J. E.D. Shah S. Bowen D.C. DeChiara Stitt T.N. Sanes J.R. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). we a different to agrin in MuSK by incubating myotubes with polyclonal antibodies directed against its that MuSK is sufficient to trigger by neuronal agrin activation of MuSK causes aggregation of AChRs and the tyrosine phosphorylation of their β-subunit with high also that AChR aggregation but not is inhibited by the of by The soluble rat agrin and have been C. Hoch W. J. Neurosci. 9: PubMed Scopus Google Scholar). of the MuSK has been and an for the extracellular part of MuSK was by the of sequence for a by a to the site of the MuSK Hopf and W. Hoch, with encoding soluble agrin of to the of and C. Cell. Biol. PubMed Scopus Google Scholar). The of and of agrin has been C. Hoch W. J. Neurosci. 9: PubMed Scopus Google Scholar). against a protein the of the domain of MuSK was by to the on was a against a to of the of MuSK by on an N-terminal MuSK in and the specificity analysis in membrane proteins a membrane of C2C12 for in was as W. 1991; PubMed Scopus Google Scholar). The M. M.A. Ullrich A. 1990; Google Scholar) was a A. Ullrich for The antibodies and and directed against the β-subunit of the AChR 1983; PubMed Scopus Google Scholar) was a J. of polyclonal antibodies against extracellular of the TGFβR and and antibodies and Fab fragments of generated by with to for at fragments and antibodies by to protein of the Fab fragments and to as as the recombinant extracellular domain of was by with expressed extracellular domain of MuSK, or the with of or Fab fragments of these antibodies for at antibodies by and antibodies with by the of Fab the concentration of was that the against the in this this Fab fragments also a the extracellular domain of MuSK as the of or on was by C2C12 containing or Fab fragments as by incubation for at in and with by to C2C12 as (7Ferns M. Hoch W. Campanelli J.T. Rupp F. Hall Z.W. Scheller R.H. Neuron. 1992; 8: 1079-1086Abstract Full Text PDF PubMed Scopus (191) Google Scholar). to in in for They to in for to with agrin or antibodies in of MuSK with and of AChRs by binding to by incubation with has been proteins by on and to membrane with antibodies and the with and with antibodies. experiments antibodies. was by analysis of To the of the or to myotubes with a of or of in for to was with the of for at C2C12 myotubes on in with or agrin for AChRs with and the of AChR in at was as C. Hoch W. J. Neurosci. 9: PubMed Scopus Google Scholar). AChR of was induced with These not in only in In experiments with or the with these as experiments The of AChR is as the of of the was by analysis dimerization is an essential for activation of receptor tyrosine kinases and in is sufficient to activate these kinases (29Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4593) Google Scholar, 30Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1291) Google Scholar, Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). therefore to and activate MuSK in the absence of agrin a directed against the of the MuSK antibodies this a in with a MuSK of was by these antibodies in a membrane of the muscle cell line C2C12 a of these antibodies was able to with MuSK antibodies as as Fab fragments to but not to MuSK was concentrated in on the surface of by incubation with to to antibodies or Fab fragments not This a for MuSK to In a of we the of these antibodies to induce tyrosine phosphorylation of MuSK by incubating C2C12 myotubes for this MuSK was of the a protein directed against its of the MuSK molecule on tyrosine was by of with antibodies. of myotubes with two of induced tyrosine phosphorylation of the kinase Several experiments that phosphorylation was phosphorylation was in the of a of the against which the been high of antibodies directed against an of MuSK did not phosphorylation of Fab fragments antibodies no although of as with and data not This that of the antibodies was necessary to induce phosphorylation of MuSK, that was able to the kinase. the with antibodies demonstrated that in the of MuSK protein that been did not account for the in tyrosine phosphorylation. Agrin induces the tyrosine phosphorylation of the β-subunit of the AChR in and C2C12 myotubes (18Wallace B.G. Qu Z. Huganir R.L. Neuron. 1991; 6: 869-878Abstract Full Text PDF PubMed Scopus (213) Google Scholar, M. M. Hall Z. J. Cell Biol. 1996; PubMed Scopus Google Scholar). therefore whether dimerization of MuSK effects. AChRs of myotubes with or Antibody-induced dimerization of MuSK a and in tyrosine phosphorylation of the AChR β-subunit that dimerization of MuSK induced not only kinase but also the phosphorylation of a able to induce AChR Fab fragments or antibodies no of the with a monoclonal directed against the β-subunit that of AChR the in we whether activation of MuSK is sufficient to induce not only phosphorylation but also of C2C12 myotubes with or with soluble agrin (4Hoch W. Ferns M. Campanelli J.T. Hall Z.W. Scheller R.H. Neuron. 1993; 11: 479-490Abstract Full Text PDF PubMed Scopus (179) Google Scholar, W. Campanelli J.T. Scheller R.H. J. Cell Biol. 1994; 126: 1-4Crossref PubMed Scopus (47) Google for AChRs with and their Agrin the of AChRs into on the surface of myotubes antibodies able to trigger a aggregation in the absence of agrin myotubes AChR The of antibodies was antibodies against an extracellular of which the neuromuscular junction in mouse muscle C. F. P. M. F. J. Neurosci. 1996; 8: PubMed Scopus Google Scholar), did not induce a of AChR directed against TGFβR and a of MuSK also no not agrin induced AChR in on C2C12 myotubes In to these aggregates, treatment induced that not in analysis of AChR The of was and of antibodies directed against extracellular of and TGFβR I, an receptor protein of the muscle no on the of of the of in MuSK and AChR phosphorylation dimerization of the MuSK molecule was required for AChR Fab fragments of did not induce AChR aggregation The experiments demonstrated that MuSK activation by agrin. However, not the that of agrin with components of the myotube surface not to MuSK a role in AChR aggregation. To a for the of of it was important to the of agrin and antibodies to induce different C2C12 myotubes with of and (4Hoch W. Ferns M. Campanelli J.T. Hall Z.W. Scheller R.H. Neuron. 1993; 11: 479-490Abstract Full Text PDF PubMed Scopus (179) Google W. Campanelli J.T. Scheller R.H. J. Cell Biol. 1994; 126: 1-4Crossref PubMed Scopus (47) Google Scholar) or with of the cell MuSK was AChRs In both tyrosine phosphorylation by the two was by In the concentration used in this induced a of MuSK phosphorylation AChR phosphorylation was with agrin induced a of MuSK phosphorylation as but required to the of agrin to AChR phosphorylation. of required to induce AChR aggregation in with MuSK phosphorylation not a activation of receptors by agrin can at a role in the agrin pathway. The of AChR aggregation in the absence of agrin to a for the of a of as as agrin-induced of AChRs B.G. J. Neurosci. 1990; 10: PubMed Google J. Neurosci. 9: PubMed Google Scholar). This in studies at an of the by which MuSK activation AChR aggregation. to a of agrin isoforms C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Abstract Full Text Full Text PDF PubMed Google Scholar, 22Gesemann M. Cavalli V. Denzer A.J. Brancaccio A. Schumacher B. Ruegg M.A. Neuron. 1996; 16: 755-767Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, J.T. Scheller R.H. 1996; PubMed Google Scholar), this binding to agrin only for part of its effects. we that as an at an additional in the agrin pathway C. Hoch W. J. Neurosci. 9: PubMed Scopus Google Scholar), which has not been To this of we whether AChR aggregation induced by agrin or antibodies. of the of AChR induced by a agrin by C. Hoch W. J. Neurosci. 9: PubMed Scopus Google Scholar). Upon inducing AChR aggregation by incubation of myotubes with we an in the of AChR that the this is not of we whether phosphorylation of MuSK and AChRs to a as it AChR aggregation. C2C12 myotubes with a agrin or MuSK antibodies in the of different of we MuSK by incubation with protein and AChRs by to and their MuSK AChR phosphorylation was by the of results with of agrin and not These experiments additional that In addition, as the that AChR aggregation but not its phosphorylation. The of was to specify the role of MuSK in the of the postsynaptic induced by agrin. have that incubation of myotubes with antibodies against MuSK the tyrosine phosphorylation of we have demonstrated that this is sufficient to induce as treatment of myotubes with agrin; AChRs to and their β-subunits became phosphorylated on tyrosine residues. Fab fragments of did not trigger although to MuSK to a as antibodies in no for binding of antibodies to other cell surface proteins the of the against which antibodies are directed it that at a to on these we that the AChR aggregation is by a dimerization of MuSK and be to of this In with antibodies different of MuSK phosphorylation AChR phosphorylation and aggregation. These indicate the of a hypothetical signal, which is by agrin but not by the antibodies. However, in a of the ligand specificity of agrin-induced no was for the of a the of AChR phosphorylation and aggregation in experiments was due to a of the agrin receptor in response to but not agrin-induced MuSK dimerization an activation of the kinase domain of MuSK the of activation of MuSK has not been a and activation has been for other receptor tyrosine the receptor J. Biol. Chem. Full Text PDF PubMed Google Scholar). this was in the activation of MuSK by a J. C. A. 15: PubMed Scopus Google Scholar). In with this activation was sufficient to trigger the phosphorylation and aggregation of although these not In to antibodies directed against the of MuSK, antibodies directed against unknown of the extracellular domain of MuSK the activation of the kinase. of these antibodies was not a different of activation B. 15: PubMed Scopus (4) Google Scholar). The of activation of MuSK that antibodies are a for role in the agrin pathway. The of results with of other to activate MuSK D.J. E.D. Shah S. Bowen D.C. DeChiara Stitt T.N. Sanes J.R. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) a between MuSK and other receptor tyrosine the role of the extracellular domain of D.J. E.D. Shah S. Bowen D.C. DeChiara Stitt T.N. Sanes J.R. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) a receptor of the extracellular domain of and the domain of MuSK with and induced phosphorylation of the and the However, activation of did not to the aggregation of AChR on the surface of C2C12 myotubes the kinase of MuSK account for AChR aggregation. In to the dimerization of the MuSK molecule and of proteins was able to induce not only the phosphorylation of AChRs but also their aggregation. This at an essential role of the extracellular domain of organizing has been suggested by experiments in a quail cell line (31Gillespie S.K.H. Balasubramanian S. Fung E.T. Huganir R.L. Neuron. 1996; 16: 953-962Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, E.D. Glass D.J. Moscoso L.M. Yancopoulos G.D. Sanes J.R. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). In this a of the MuSK (31Gillespie S.K.H. Balasubramanian S. Fung E.T. Huganir R.L. Neuron. 1996; 16: 953-962Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and a MuSK in which most of the domain been (32Apel E.D. Glass D.J. Moscoso L.M. Yancopoulos G.D. Sanes J.R. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar) by In muscle a of rat MuSK AChR D.J. E.D. Shah S. Bowen D.C. DeChiara Stitt T.N. Sanes J.R. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the of tyrosine kinase for this data the that two are necessary to induce the aggregation of AChRs in myotubes (32Apel E.D. Glass D.J. Moscoso L.M. Yancopoulos G.D. Sanes J.R. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, Yancopoulos G.D. Curr. Opin. PubMed Scopus Google Scholar); the is the kinase of MuSK, and the appears to the of other proteins with It is in which this on MuSK activation which proteins with The most likely appears to be RATL, which rapsyn and the AChRs to MuSK (32Apel E.D. Glass D.J. Moscoso L.M. Yancopoulos G.D. Sanes J.R. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). the of induced by MuSK dimerization be important for AChR aggregation. This appears data that a activation of by the binding of agrin is not essential for this pathway. that be by the binding of agrin to can be by the dimerization of of of the agrin pathway have been an of tyrosine both phosphorylation and aggregation of AChRs (17Wallace B.G. J. Cell Biol. 1994; 125: 661-668Crossref PubMed Scopus (99) Google Scholar) and the in the pathway data suggest that a of which with the Heparin treatment a it inhibited AChR aggregation induced by a agrin C. Hoch W. J. Neurosci. 9: PubMed Scopus Google Scholar) and by antibodies by it did not affect the phosphorylation of MuSK or This with receptor aggregation be a exclusively with the in the agrin pathway. The extracellular domain of MuSK, which is in this is to and other into the The protein with has not been identified but is an The of and of the agrin pathway be in the to the and with the identified and for Ullrich and for the of and and for of the also to for