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

Elmar Porten,Beate Seliger,Verena A Schneider,Stefan Wöll,Daniela Stangel,Rene Ramseger,Stephan Kröger

doi:10.1074/jbc.m109.039420

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 beta-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.

Highlights

Little is known about the role of agrin in tissues other than skeletal muscle, in particular in the central nervous system
Our results show that an aspartic acid residue within the seventh follistatin-like domain is essential for process induction
These results demonstrate that the C-terminal half of TM-agrin is not required for process induction, and, the region involved in process induction and the domains responsible for the synaptogenic role of agrin at the neuromuscular junction are localized within different parts of the agrin protein

Summary

EXPERIMENTAL PROCEDURES

Animals—Fertile White Leghorn (Gallus gallus domesticus) chicken eggs were purchased from a local hatchery and incubated at 38 °C in a humid atmosphere. Expression Constructs—The cDNA sequence for chick TMagrin has been described elsewhere [18] Deletion constructs from these sequences were generated using the PCR and Phusion high fidelity DNA-polymerase (BioCat, Heidelberg, Germany). The constructs were cloned via EcoRI and XbaI or via HindIII and EcoRI restriction sites into the pcDNA4TO expression vector (Invitrogen). To obtain a GFP tag at the C terminus the TMFD8 and TMFD6 constructs were cloned via EcoRI and BamHI restriction sites into the pEGFP-N1 expression vector (Clontech, Mountain View, CA) using the following primers: TMFD8GFP: 5Ј-GTGTGGTGGAATTCACAGCATGACGGC-3Ј (sense) and 5Ј-CGCGGATCCGTCTGACTGCAGTGC-3Ј (antisense); and TMFD6-GFP: 5Ј-GTGTGGTGGAATTCACAGCATGACGGC-3Ј (sense) and 5Ј-CGCGGATCCTCATAGGTGAGGCCATCG-3Ј (antisense). To generate the constructs with an altered order of the follistatin-like domains, an intermediate called TMFD6 –503 was created and cloned via HindIII and EcoRI restriction sites into the pEGFP-N1 vector. The following primers were used: TMFD6 –503: 5Ј-ATAAAGCTTAGCATGACGGCTTGCCAGTACC-3Ј (sense) and 5Ј-TAGAATTCTTGCAGTCGCCTTGGGCAGC-3Ј (antisense); TMFD6ϩFD1-GFP: 5Ј-ATGAATTCAGATGCCTGCCGAGGGATGC-3Ј (sense) and 5Ј-ATGGATCCGTGCTGCCAAAGCTGC-3Ј (antisense); TMFD6ϩFD6-GFP: 5Ј-ATGAATTCCGACCGCTGTGGCAAGTGC-3Ј (sense) and 5Ј-ATGGATCCAAGGAGCACACCGTCGTGC-3Ј (antisense); and TMFD6ϩFD8-GFP: 5Ј-ATGAATTCTGAGGACGAATGTGGCTCAGG-3Ј (sense) and 5Ј-CGCGGATCCGTCTGACTGCAGTGC-3Ј (antisense)

To generate the constructs containing successively less

RESULTS

DISCUSSION