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 ␤-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.Agrin is a proteoglycan with a molecular mass of Ͼ500 kDa that is expressed in many tissues (1, 2). 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 (2-4). Accordingly, mice with an inactivation of the agrn gene die at birth due to non-functional neuromuscular junctions and consequent respiratory failure (5).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. 1, 2, 6). Although neurons from mice with a targeted deletion of the agrn gene form synaptic specializations in vitro and in vivo (7,8), the acute suppression of agrin expression or function by antisense oligonucleotides or antibodies influences the formation and function of interneuronal synapses (9, 10). 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 3 (11). 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 lamini...

The role of agrin in synaptic development, plasticity and signaling in the central nervous system

“…Likewise, induction of filopodia in neurons or filopodia-like processes in non-neuronal cells by overexpression of Tm-agrin is also independent of Z-site inserts (McCroskery et al, 2006). In fact, this latter activity is dependent on the N-terminal moiety of Tm-agrin, importantly including the transmembrane domain (McCroskery et al, 2006) and 3 of the 8 follistatin-like domains (fig.1) in the N-terminal extracellular moiety (Porten et al, 2010). There is also strong evidence that the glycosaminoglycan (GAG) chains (fig.…”

Induction of filopodia-like protrusions by transmembrane agrin: Role of agrin glycosaminoglycan chains and Rho-family GTPases

“…In this respect, it is interesting that in the present study, the elimination of the HSPG GAG chains, which are inserted between the 7th and 8th follistatin domains, more strongly reduced BRF formation than elimination of the CS GAG chains. However, the following differences between the assays and constructs used by Porten et al [51] and in the present study, leave room for interpretation as to the specific roles of the GAG chains and follistatin domains. First, the present study used an assay in which the portion of the total cell area occupied by BRFs was assayed.…”

“…Similar protrusions were observed in COS-1 cells overexpressing the proteoglycan syndecan-2 [50]. Recently, formation of filopodia-like processes identical in appearance to BRFs was described in TM-agrin transfected COS7 and HEK293 cells [51]. Filopodia, microspikes and retraction fibers contain actin filament bundles and are regarded as a continuum of structures distinguished by the protrusive activity of the surrounding lamellipodia [44].…”

“…In a recent article (Porten et al, 2009) [51], it was reported that N-terminal follistatin domains, particularly the 7th follistatin domain of TM-agrin, were necessary and sufficient to induce the formation of filopodia-like processes in HEK293 cells and cultured embryonic chick tectal neurons. When the 3 serines that serve as HSPG GAG chain attachment sites were mutated, the N-terminal construct lost 70% of its process-inducing activity but when the domain containing those attachment sites as well as the 8th follistatin domain was deleted, full activity was restored.…”

Emerging roles for MAP kinases in agrin signaling

“…Antibody-induced clustering or overexpression of transmembrane agrin in neuronal and non-neuronal cells induces filopodia. [40][41][42][43] This activity depends on domains in the N-terminal third of the agrin protein, 43,44 not overlapping with the C-terminal domains responsible for AChR clustering. ERK1/2 is likely to mediate filopodia induction by transmembrane agrin as PD98059, a MEK1/2 inhibitor, 45 blocked this activity.…”