The Process-inducing Activity of Transmembrane Agrin Requires Follistatin-like Domains

Abstract

Clustering or overexpression of the transmembrane form of the extracellular matrix proteoglycan agrin in neurons results in the formation of numerous highly motile filopodia-like processes extending from axons and dendrites. Here we show that similar processes can be induced by overexpression of transmembrane-agrin in several non-neuronal cell lines. Mapping of the process-inducing activity in neurons and non-neuronal cells demonstrates that the cytoplasmic part of transmembrane agrin is dispensable and that the extracellular region is necessary for process formation. Site-directed mutagenesis reveals an essential role for the loop between β-sheets 3 and 4 within the Kazal subdomain of the seventh follistatin-like domain of TM-agrin. An aspartic acid residue within this loop is critical for process formation. The seventh follistatin-like domain could be functionally replaced by the first and sixth but not by the eighth follistatin-like domain, demonstrating a functional redundancy among some follistatin-like domains of agrin. Moreover, a critical distance of the seventh follistatin-like domain to the plasma membrane appears to be required for process formation. These results demonstrate that different regions within the agrin protein are responsible for synapse formation at the neuromuscular junction and for process formation in central nervous system neurons and suggest a role for agrin’s follistatin-like domains in the developing central nervous system. Clustering or overexpression of the transmembrane form of the extracellular matrix proteoglycan agrin in neurons results in the formation of numerous highly motile filopodia-like processes extending from axons and dendrites. Here we show that similar processes can be induced by overexpression of transmembrane-agrin in several non-neuronal cell lines. Mapping of the process-inducing activity in neurons and non-neuronal cells demonstrates that the cytoplasmic part of transmembrane agrin is dispensable and that the extracellular region is necessary for process formation. Site-directed mutagenesis reveals an essential role for the loop between β-sheets 3 and 4 within the Kazal subdomain of the seventh follistatin-like domain of TM-agrin. An aspartic acid residue within this loop is critical for process formation. The seventh follistatin-like domain could be functionally replaced by the first and sixth but not by the eighth follistatin-like domain, demonstrating a functional redundancy among some follistatin-like domains of agrin. Moreover, a critical distance of the seventh follistatin-like domain to the plasma membrane appears to be required for process formation. These results demonstrate that different regions within the agrin protein are responsible for synapse formation at the neuromuscular junction and for process formation in central nervous system neurons and suggest a role for agrin’s follistatin-like domains in the developing central nervous system. IntroductionAgrin is a proteoglycan with a molecular mass of >500 kDa that is expressed in many tissues (1Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 295-308Crossref PubMed Scopus (263) Google Scholar, 2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar). The function of agrin is best characterized in skeletal muscle where it is a key organizer during formation, maintenance, and regeneration of the neuromuscular junction (2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar, 3Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (805) Google Scholar, 4Lin S. Landmann L. Ruegg M.A. Brenner H.R. J. Neurosci. 2008; 28: 3333-3340Crossref PubMed Scopus (55) Google Scholar). Accordingly, mice with an inactivation of the agrn gene die at birth due to non-functional neuromuscular junctions and consequent respiratory failure (5Gautam M. Noakes P.G. Moscoso L. Rupp F. Scheller R.H. Merlie J.P. Sanes J.R. Cell. 1996; 85: 525-535Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar).Little is known about the role of agrin in tissues other than skeletal muscle, in particular in the central nervous system (for review see Refs. 1Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 295-308Crossref PubMed Scopus (263) Google Scholar, 2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar, 6Kröger S. Schröder J.E. News Physiol. Sci. 2002; 17: 207-212PubMed Google Scholar). Although neurons from mice with a targeted deletion of the agrn gene form synaptic specializations in vitro and in vivo (7Li Z. Hilgenberg L.G. O'Dowd D.K. Smith M.A. J. Neurobiol. 1999; 39: 547-557Crossref PubMed Scopus (55) Google Scholar, 8Serpinskaya A.S. Feng G. Sanes J.R. Craig A.M. Dev. Biol. 1999; 205: 65-78Crossref PubMed Scopus (102) Google Scholar), the acute suppression of agrin expression or function by antisense oligonucleotides or antibodies influences the formation and function of interneuronal synapses (9Ferreira A. J. Cell Sci. 1999; 112: 4729-4738Crossref PubMed Google Scholar, 10Böse C.M. Qiu D. Bergamaschi A. Gravante B. Bossi M. Villa A. Rupp F. Malgaroli A. J. Neurosci. 2000; 20: 9086-9095Crossref PubMed Google Scholar). Likewise, brains of agrin-deficient mice, whose perinatal death was prevented by the re-expression of agrin in motor neurons, have a severely reduced number of pre- and postsynaptic specializations as well as functional deficits at excitatory synapses in the CNS 3The abbreviations used are: CNScentral nervous systemEembryonic dayLRP4low density lipoprotein-like receptor 4TM-agrintransmembrane form of agrinHEKhuman embryonic kidney cellGAGglycosaminoglycanEGFepidermal growth factorGFPgreen fluorescent proteinNtA-agrinNtA domain-containing agrin isoform. (11Ksiazek I. Burkhardt C. Lin S. Seddik R. Maj M. Bezakova G. Jucker M. Arber S. Caroni P. Sanes J.R. Bettler B. Ruegg M.A. J. Neurosci. 2007; 27: 7183-7195Crossref PubMed Scopus (98) Google Scholar). Although these data are consistent with a role of agrin during CNS synaptogenesis, the precise function of agrin during CNS development remains unclear.Agrin has been cloned from several species, and the sequences are highly homologous. The agrin cDNAs predict a number of domains with similarity to other extracellular matrix proteins, including four EGF-like repeats and three domains with similarity to globular domain of the laminin α-chain (LG domain) within the C-terminal half of the protein. In skeletal muscle fibers the C-terminal LG domains of agrin bind to α-dystroglycan (12Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.R. Neuron. 1994; 12: 1173-1180Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 13Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Abstract Full Text Full Text PDF PubMed Google Scholar, 14Gesemann M. Brancaccio A. Schumacher B. Ruegg M.A. J. Biol. Chem. 1998; 273: 600-605Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and to the low density lipoprotein receptor-like protein 4 (LRP4 (15Zhang B. Luo S. Wang Q. Suzuki T. Xiong W.C. Mei L. Neuron. 2008; 60: 285-297Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 16Kim N. Stiegler A.L. Cameron T.O. Hallock P.T. Gomez A.M. Huang J.H. Hubbard S.R. Dustin M.L. Burden S.J. Cell. 2008; 135: 334-342Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar)). The binding of agrin to LRP4 activates the tyrosine kinase MuSK and initiates a complex intracellular signaling cascade resulting in the formation of most if not all pre- and postsynaptic specializations (1Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 295-308Crossref PubMed Scopus (263) Google Scholar, 2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar). The functions of the other domains, particularly of the 9 follistatin-like domains within the N-terminal half of the agrin protein are unknown.Alternative first exon usage generates either a secreted soluble agrin molecule (NtA-agrin) or a transmembrane form of agrin (TM-agrin (17Burgess R.W. Skarnes W.C. Sanes J.R. J. Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (139) Google Scholar, 18Neumann F.R. Bittcher G. Annies M. Schumacher B. Kröger S. Ruegg M.A. Mol. Cell. Neurosci. 2001; 17: 208-225Crossref PubMed Scopus (72) Google Scholar)). The secreted form of agrin binds to the laminin γ1-chain via its NtA domain resulting in stable association of this agrin isoform with basal R. M. M. Ruegg M.A. J. Cell Biol. PubMed Scopus Google Scholar). In in the NtA domain is replaced by a that agrin a transmembrane protein and the agrin protein in in the plasma membrane (17Burgess R.W. Skarnes W.C. Sanes J.R. J. Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (139) Google Scholar, 18Neumann F.R. Bittcher G. Annies M. Schumacher B. Kröger S. Ruegg M.A. Mol. Cell. Neurosci. 2001; 17: 208-225Crossref PubMed Scopus (72) Google is expressed axons and of CNS neurons M. Kröger S. Mol. Cell. Neurosci. 2002; 20: PubMed Scopus Google Scholar, M. Bittcher G. R. J. S. C. M.A. Kröger S. Mol. Cell. Neurosci. PubMed Scopus Google Scholar, S. A. Lin L. Mol. Cell. Neurosci. PubMed Scopus Google Scholar), and it has been that as a receptor or M. Bittcher G. R. J. S. C. M.A. Kröger S. Mol. Cell. Neurosci. PubMed Scopus Google Scholar). In with this it was that or overexpression of in neurons during the of growth the and the formation of numerous filopodia-like processes extending from the M. Bittcher G. R. J. S. C. M.A. Kröger S. Mol. Cell. Neurosci. PubMed Scopus Google Scholar, S. A. Lin L. Mol. Cell. Neurosci. PubMed Scopus Google Scholar). The formation of these processes is by the of a complex signaling as well as the of protein and S. A. Lin L. Mol. Cell. Neurosci. PubMed Scopus Google Scholar, R. R. Kröger S. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). The function of the filopodia-like processes in the developing CNS is but during the formation of excitatory synapses A. D. S. J. J. T. B. P. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). with this mice that CNS agrin synapses (11Ksiazek I. Burkhardt C. Lin S. Seddik R. Maj M. Bezakova G. Jucker M. Arber S. Caroni P. Sanes J.R. Bettler B. Ruegg M.A. J. Neurosci. 2007; 27: 7183-7195Crossref PubMed Scopus (98) Google this we demonstrate that overexpression of in non-neuronal cell can processes similar to induced in CNS that the process-inducing activity of is not to the for the process-inducing activity of by the region necessary for this results show that an aspartic acid residue within the seventh follistatin-like domain is essential for process particular acid is highly in other follistatin-like domains of TM-agrin. Accordingly, the seventh follistatin-like domain could functionally be replaced by the first or sixth follistatin-like These data show that follistatin-like domains are critical for process formation and a role for as a receptor or in the developing of this was to the for the formation of filopodia-like results demonstrate three for process-inducing An association of with the plasma the of the loop between 3 and 4 within the seventh follistatin-like domain, and a critical distance of the follistatin-like domain to the plasma domains are of extracellular matrix proteins, including for P. R. J. PubMed Scopus Google Scholar, J.E. M.A. J. Cell. PubMed Scopus Google Scholar), and S.J. J. PubMed Scopus Google Scholar), C. S. M. S. H. M. P. J. 1999; PubMed Scopus (55) Google Scholar), and C. N. N. S. C. M. P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, C. S. M. P. J. 2003; PubMed Scopus Google Scholar), and T. T. M. H. A. M. C. 1999; PubMed Scopus Google Scholar). Although follistatin-like domains are in a number of extracellular matrix proteins, is known about that follistatin-like different functions P. B. Q. Rev. 1996; PubMed Google Scholar). show that follistatin-like domain of agrin is essential for the formation of filopodia-like processes expressed in neurons and non-neuronal Moreover, the expression of can processes for in cells or the appears to be a molecular for process In with this is the that the formation of the processes is the of the of a signaling cascade that of and membrane R. R. Kröger S. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). are signaling with functions during CNS development and synapse formation (for review see Refs. D. Nat. Rev. Mol. Cell Biol. 2000; PubMed Scopus Google Scholar, Nat. Rev. Neurosci. 2007; PubMed Scopus Google Scholar). The of in all cells the formation of processes of a of cell from different Likewise, the of the the formation of filopodia-like processes in to overexpression of several other proteins, including for the transmembrane proteoglycan A. J.R. Biol. 2003; PubMed Scopus Google Scholar), R. Mol. Cell. Neurosci. 2002; PubMed Scopus Google Scholar), or M.A. J. Cell Biol. 2000; PubMed Scopus Google of the domain of M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) or of a of the first follistatin-like domains from P. R. J. PubMed Scopus Google Scholar) have a by the These follistatin-like domains an N-terminal EGF-like subdomain characterized by via a and a C-terminal part with similarity to the of of three β-sheets and an P. R. J. PubMed Scopus Google Scholar, M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google see for the of results show that the between β-sheets 3 and 4 is critical for the formation of the filopodia-like processes in neurons and non-neuronal region has function in follistatin-like the between the domain and the follistatin-like domain within is by the P. R. J. PubMed Scopus Google Scholar). Likewise, the binding of to not is to the N-terminal between β-sheets and of the first domain of S. N. S. PubMed Scopus Google Scholar, S. S. N. S. PubMed Scopus Google Scholar). The the consistent with to the M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Q. A.L. 2000; PubMed Scopus Google Scholar). the other in the follistatin-like domain of an residue between 3 and 4 in has been to be to P. R. J. PubMed Scopus Google Scholar). this is not in of the follistatin-like domains of that at this is not to process number of functions of agrin have been to the and W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), including with low molecular growth growth W. J. Neurobiol. 2003; PubMed Scopus Google Scholar) or of growth W. J. PubMed Scopus Google Scholar). results show that are dispensable for the formation of filopodia-like processes in neurons and non-neuronal an of growth or signaling and by agrin process formation. all three that as between follistatin-like domains and W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) to the protein a reduced process-inducing deletion of the eighth follistatin-like domain with the be that the have a role in the of the eighth follistatin-like In the of the eighth follistatin-like domain an and with the function of follistatin-like domain not be in the of the and follistatin-like domain the process-inducing activity of the follistatin-like domain could be functionally replaced by domains and the eighth follistatin-like domain could not for the seventh the eighth follistatin-like domain of the aspartic acid residue between 3 and it from follistatin-like domains and in at an between and and the at its for a of the different follistatin-like domains of the acid between β-sheets and is in the EGF-like is not in process formation, the for the failure of the eighth follistatin-like domain to functionally for follistatin-like domain is the of the Although we have not the of the by the of the eighth follistatin-like domain a molecular mass of highly in for as well as in W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, W. J. PubMed Scopus Google Scholar). The of this in the eighth follistatin-like domain that the eighth follistatin-like domain have in the the the of the follistatin-like domain with other or with the to follistatin-like domains, of are as an by demonstrates a of the follistatin-like domains in agrin T. C. Schumacher B. J. Ruegg M.A. J. 1998; 17: PubMed Scopus Google Scholar). has been for the of the of follistatin-like domains P. R. J. PubMed Scopus Google Scholar). that the of the N-terminal part of follistatin-like domain with the of the follistatin-like domain P. R. J. PubMed Scopus Google Scholar). the loop between 3 and 4 is part of the C-terminal it is that in this region the between follistatin-like the process-inducing activity of follistatin-like domain is not by the deletion of the eighth follistatin-like the of a C-terminal appears not to be necessary for process filopodia-like processes are known to as for synapses during development as well as during synapse (for review see Refs. R. T. Nat. Rev. Neurosci. PubMed Scopus Google Scholar, L. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). the can synapses with a Smith S.J. Neuron. 1996; 17: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Neurosci. 1998; PubMed Google Scholar, G. A. Rev. 2008; PubMed Scopus Google Scholar). the number of functional synapses is of the within and a consistent with a and a of these processes C. T. Neuron. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). processes and the filopodia-like processes induced by are similar with to of and and to intracellular signaling Smith S.J. Neuron. 1996; 17: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Neurosci. 1998; PubMed Google Scholar, Smith S.J. J. Neurosci. 1996; PubMed Google Scholar, Z. A. R. 2000; PubMed Scopus Google Scholar, A. L. Nat. Neurosci. PubMed Scopus Google Scholar). similarity that be in the formation of synapses in the developing CNS by the formation of filopodia-like processes dendrites. with this of reduced the number of synapses in neurons S. A. Lin L. 2009; PubMed Scopus Google Scholar). In mice that agrin in the CNS have a in the number of excitatory synapses and in the (11Ksiazek I. Burkhardt C. Lin S. Seddik R. Maj M. Bezakova G. Jucker M. Arber S. Caroni P. Sanes J.R. Bettler B. Ruegg M.A. J. Neurosci. 2007; 27: 7183-7195Crossref PubMed Scopus (98) Google interneuronal synapses a that is in the and in the synaptic has been to A. P. S. T. P. B. P. J. 2008; PubMed Scopus Google Scholar). of agrin by at within the agrin a plasma protein as well as soluble from the with molecular of and R. S. S. B. C. J. D. A. A. S. A. B. B. P. J. 2007; PubMed Scopus Google Scholar). to synapses as well as of agrin the and postsynaptic activity and acid receptor as well as A. D. S. J. J. T. B. P. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. M. A. P. J. Neurosci. 2008; 28: PubMed Scopus Google Scholar). mice form filopodia-like processes A. D. S. J. J. T. B. P. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar), demonstrating that agrin is required for process formation. The C-terminal agrin by is to filopodia-like processes to and the is to the in filopodia-like processes in mice A. D. S. J. J. T. B. P. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). overexpression of as well as of the soluble C-terminal agrin by can the formation of and process formation and interneuronal synapse formation. is that the predict that binding of the C-terminal agrin to or of to the formation of filopodia-like processes for by of and be a receptor or for the binding of the agrin by of the tyrosine kinase MuSK (11Ksiazek I. Burkhardt C. Lin S. Seddik R. Maj M. Bezakova G. Jucker M. Arber S. Caroni P. Sanes J.R. Bettler B. Ruegg M.A. J. Neurosci. 2007; 27: 7183-7195Crossref PubMed Scopus (98) Google Scholar), of the agrin receptor LRP4 (15Zhang B. Luo S. Wang Q. Suzuki T. Xiong W.C. Mei L. Neuron. 2008; 60: 285-297Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 16Kim N. Stiegler A.L. Cameron T.O. Hallock P.T. Gomez A.M. Huang J.H. Hubbard S.R. Dustin M.L. Burden S.J. Cell. 2008; 135: 334-342Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar), or of the L.G. H. H. O'Dowd D.K. Smith M.A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) all of are expressed at synapses in the In the results in this demonstrate a critical of the seventh follistatin-like domain within in the formation of filopodia-like IntroductionAgrin is a proteoglycan with a molecular mass of >500 kDa that is expressed in many tissues (1Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 295-308Crossref PubMed Scopus (263) Google Scholar, 2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar). The function of agrin is best characterized in skeletal muscle where it is a key organizer during formation, maintenance, and regeneration of the neuromuscular junction (2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar, 3Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (805) Google Scholar, 4Lin S. Landmann L. Ruegg M.A. Brenner H.R. J. Neurosci. 2008; 28: 3333-3340Crossref PubMed Scopus (55) Google Scholar). Accordingly, mice with an inactivation of the agrn gene die at birth due to non-functional neuromuscular junctions and consequent respiratory failure (5Gautam M. Noakes P.G. Moscoso L. Rupp F. Scheller R.H. Merlie J.P. Sanes J.R. Cell. 1996; 85: 525-535Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar).Little is known about the role of agrin in tissues other than skeletal muscle, in particular in the central nervous system (for review see Refs. 1Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 295-308Crossref PubMed Scopus (263) Google Scholar, 2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar, 6Kröger S. Schröder J.E. News Physiol. Sci. 2002; 17: 207-212PubMed Google Scholar). Although neurons from mice with a targeted deletion of the agrn gene form synaptic specializations in vitro and in vivo (7Li Z. Hilgenberg L.G. O'Dowd D.K. Smith M.A. J. Neurobiol. 1999; 39: 547-557Crossref PubMed Scopus (55) Google Scholar, 8Serpinskaya A.S. Feng G. Sanes J.R. Craig A.M. Dev. Biol. 1999; 205: 65-78Crossref PubMed Scopus (102) Google Scholar), the acute suppression of agrin expression or function by antisense oligonucleotides or antibodies influences the formation and function of interneuronal synapses (9Ferreira A. J. Cell Sci. 1999; 112: 4729-4738Crossref PubMed Google Scholar, 10Böse C.M. Qiu D. Bergamaschi A. Gravante B. Bossi M. Villa A. Rupp F. Malgaroli A. J. Neurosci. 2000; 20: 9086-9095Crossref PubMed Google Scholar). Likewise, brains of agrin-deficient mice, whose perinatal death was prevented by the re-expression of agrin in motor neurons, have a severely reduced number of pre- and postsynaptic specializations as well as functional deficits at excitatory synapses in the CNS 3The abbreviations used are: CNScentral nervous systemEembryonic dayLRP4low density lipoprotein-like receptor 4TM-agrintransmembrane form of agrinHEKhuman embryonic kidney cellGAGglycosaminoglycanEGFepidermal growth factorGFPgreen fluorescent proteinNtA-agrinNtA domain-containing agrin isoform. (11Ksiazek I. Burkhardt C. Lin S. Seddik R. Maj M. Bezakova G. Jucker M. Arber S. Caroni P. Sanes J.R. Bettler B. Ruegg M.A. J. Neurosci. 2007; 27: 7183-7195Crossref PubMed Scopus (98) Google Scholar). Although these data are consistent with a role of agrin during CNS synaptogenesis, the precise function of agrin during CNS development remains unclear.Agrin has been cloned from several species, and the sequences are highly homologous. The agrin cDNAs predict a number of domains with similarity to other extracellular matrix proteins, including four EGF-like repeats and three domains with similarity to globular domain of the laminin α-chain (LG domain) within the C-terminal half of the protein. In skeletal muscle fibers the C-terminal LG domains of agrin bind to α-dystroglycan (12Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.R. Neuron. 1994; 12: 1173-1180Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 13Hopf C. Hoch W. J. Biol. Chem. 1996; 271: 5231-5236Abstract Full Text Full Text PDF PubMed Google Scholar, 14Gesemann M. Brancaccio A. Schumacher B. Ruegg M.A. J. Biol. Chem. 1998; 273: 600-605Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and to the low density lipoprotein receptor-like protein 4 (LRP4 (15Zhang B. Luo S. Wang Q. Suzuki T. Xiong W.C. Mei L. Neuron. 2008; 60: 285-297Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 16Kim N. Stiegler A.L. Cameron T.O. Hallock P.T. Gomez A.M. Huang J.H. Hubbard S.R. Dustin M.L. Burden S.J. Cell. 2008; 135: 334-342Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar)). The binding of agrin to LRP4 activates the tyrosine kinase MuSK and initiates a complex intracellular signaling cascade resulting in the formation of most if not all pre- and postsynaptic specializations (1Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 295-308Crossref PubMed Scopus (263) Google Scholar, 2Kröger S. Pfister H. Fut. Neurol. 2009; 4: 67-86Crossref Scopus (6) Google Scholar). The functions of the other domains, particularly of the 9 follistatin-like domains within the N-terminal half of the agrin protein are unknown.Alternative first exon usage generates either a secreted soluble agrin molecule (NtA-agrin) or a transmembrane form of agrin (TM-agrin (17Burgess R.W. Skarnes W.C. Sanes J.R. J. Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (139) Google Scholar, 18Neumann F.R. Bittcher G. Annies M. Schumacher B. Kröger S. Ruegg M.A. Mol. Cell. Neurosci. 2001; 17: 208-225Crossref PubMed Scopus (72) Google Scholar)). The secreted form of agrin binds to the laminin γ1-chain via its NtA domain resulting in stable association of this agrin isoform with basal R. M. M. Ruegg M.A. J. Cell Biol. PubMed Scopus Google Scholar). In in the NtA domain is replaced by a that agrin a transmembrane protein and the agrin protein in in the plasma membrane (17Burgess R.W. Skarnes W.C. Sanes J.R. J. Cell Biol. 2000; 151: 41-52Crossref PubMed Scopus (139) Google Scholar, 18Neumann F.R. Bittcher G. Annies M. Schumacher B. Kröger S. Ruegg M.A. Mol. Cell. Neurosci. 2001; 17: 208-225Crossref PubMed Scopus (72) Google is expressed axons and of CNS neurons M. Kröger S. Mol. Cell. Neurosci. 2002; 20: PubMed Scopus Google Scholar, M. Bittcher G. R. J. S. C. M.A. Kröger S. Mol. Cell. Neurosci. PubMed Scopus Google Scholar, S. A. Lin L. Mol. Cell. Neurosci. PubMed Scopus Google Scholar), and it has been that as a receptor or M. Bittcher G. R. J. S. C. M.A. Kröger S. Mol. Cell. Neurosci. PubMed Scopus Google Scholar). In with this it was that or overexpression of in neurons during the of growth the and the formation of numerous filopodia-like processes extending from the M. Bittcher G. R. J. S. C. M.A. Kröger S. Mol. Cell. Neurosci. PubMed Scopus Google Scholar, S. A. Lin L. Mol. Cell. Neurosci. PubMed Scopus Google Scholar). The formation of these processes is by the of a complex signaling as well as the of protein and S. A. Lin L. Mol. Cell. Neurosci. PubMed Scopus Google Scholar, R. R. Kröger S. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). The function of the filopodia-like processes in the developing CNS is but during the formation of excitatory synapses A. D. S. J. J. T. B. P. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). with this mice that CNS agrin synapses (11Ksiazek I. Burkhardt C. Lin S. Seddik R. Maj M. Bezakova G. Jucker M. Arber S. Caroni P. Sanes J.R. Bettler B. Ruegg M.A. J. Neurosci. 2007; 27: 7183-7195Crossref PubMed Scopus (98) Google this we demonstrate that overexpression of in non-neuronal cell can processes similar to induced in CNS that the process-inducing activity of is not to the for the process-inducing activity of by the region necessary for this results show that an aspartic acid residue within the seventh follistatin-like domain is essential for process particular acid is highly in other follistatin-like domains of TM-agrin. Accordingly, the seventh follistatin-like domain could functionally be replaced by the first or sixth follistatin-like These data show that follistatin-like domains are critical for process formation and a role for as a receptor or in the developing