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 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.

FIGURE 1.

Expression of TM-agrin induces processes in non-neuronal cells. Human embryonic kidney cells (A–D) or COS-7 cells (E and F) were transiently transfected with a cDNA coding for either NtA-agrin (A and B) or for TM-agrin (C–F). Cells were stained with Alexa594-conjugated phalloidin to reveal the actin filaments (A and C) and with anti-agrin antibodies (B, D, E, and F). Although the expression of NtA-agrin did not apparently affect the morphology of the transfected HEK293 cells (A and B), expression of TM-agrin induced the formation of numerous actin filament-containing processes by the transfected cells (C). The actin filaments extended until close to the tip of the processes (arrows in C and D). The processes did not collapse and retract but instead continued to grow in culture (A–D show cells 24 h after transfection; E and F show cells 48 h post transfection). Scale bars in A, C, E, and F: 10 μm.

FIGURE 2.

Follistatin-like domains are required for process-inducing activity. Schematic representation of the different TM-agrin constructs used to localize the region responsible for the formation of the filopodia-like processes (left). The different domains within the TM-agrin protein are specified on the bottom. The nine follistatin-like domains are numbered according to their position starting from the N terminus. Quantification of the transfected cells extending processes is shown on the right. Note that deletion of most of the extracellular part of TM-agrin has no influence on process formation. However, deletion of follistatin-like domains 7 and 8 (TM-agrin-FD6 construct) abolished process-inducing activity. The bars represent the mean ± S.E. with n = 3.

FIGURE 3.

The seventh follistatin-like domain is required for process formation. To determine the role of the heparan sulfate side chains in the formation of the filopodia-like processes, the construct TMFD8 was truncated in the region between follistatin-like domain 7 (FD7; shown in blue) and 8 (FD8, shown in red). The exact amino acid sequence containing the three serine residues (serine 573, 576, and 578, respectively) used as GAG chain attachment sites of chick TM-agrin is shown on the left. The percentage of transfected cells having filopodia-like processes is shown on the right (mean ± S.E. with n = 3). Complete deletion of the GAG side-chain attachment sites did not result in a loss of the process-inducing activity (constructs TMFD7–572 and TMFD7–568), demonstrating that the GAG side chains are dispensable for process induction. In contrast, deletion of two additional amino acids (cysteine at position 567 and glutamic acid at position 568) completely abolished process-inducing activity (construct TMFD7–566).

FIGURE 4.

The disulfide bond between cysteines 535 and 567 within the seventh follistatin-like domain is critical for process-inducing activity. To interrupt the disulfide bond between cysteine 535 and 567, both residues were individually mutated to glycine giving rise to the TMFD8 C535G and TMFD8 C567G constructs, respectively. Each mutation resulted in a loss of process-inducing activity. In contrast, mutation of the cysteine corresponding to the cysteine 535 in the neighboring sixth follistatin-like domain (construct TMFD8 C470G) or mutation of the asparagine residue to aspartic acid within the seventh follistatin-like domain (TMFC8 N544D) did not result in a loss of process-inducing activity. The bars in B show the mean ± S.E. with n = 3.

FIGURE 5.

The loop between β-sheet 3 and 4 within the seventh follistatin-like domain is critical for process formation. The amino acid sequence of follistatin-like domain 7 is shown in A. The 10 cysteine residues are shown in red, and their disulfide bonds are schematically indicated. The location of the α-helix and the five β-sheets within the follistatin-like domain are schematically indicated on top of the amino acid sequence. The mutated amino acids are shown in green and are marked by an asterisk. B shows the quantification of the process formation activity of the various mutated constructs. The values represent the mean ± S.E. with n = 3.

FIGURE 6.

Follistatin-like domain 7 can be replaced by domains 1 and 6 but not by domain 8. To determine if other follistatin-like domains of TM-agrin also have process-inducing activity when placed at the appropriate position, constructs were generated where the seventh follistatin-like domain was replaced by either the first follistatin-like domain (construct TMFD6+FD1-GFP), by the sixth follistatin-like domain (construct TMFD6+FD6-GFP), or by the eighth follistatin-like domain (construct TMFD6+FD8-GFP). Although the first and sixth follistatin-like domains can functionally substitute for the seventh follistatin-like domain, replacing follistatin-like domain 7 with the eighth follistatin-like domain resulted in a severe reduction of the number of processes extended by the transfected cells. Moreover, a critical distance of the seventh follistatin-like domain to the plasma membrane appears to be necessary for process formation, because deletion of the first and second follistatin-like domain (construct TMFD3–8-GFP) resulted in a loss of the process-formation activity, whereas deletion of only the first follistatin-like domain (construct TMFD2–8-GFP) did not influence process formation.

FIGURE 7.

Overexpression of TM-agrin mutation constructs in tectal neurons. Representative images of tectal neurons after transfection with TMFD8-GFP (A and B), the empty EGFP-N1 vector (C), TMFD8-G539P-GFP (D), TMFD8-QQ555,556EE-GFP (E), or TMFD8-C535G-GFP (F) are shown. Tectal neurons were stained 3 days after transfection with Alexa594-conjugated phalloidin (A, C, D, and E) or with anti-GFP antibodies (B and F). When TMFD8 or TMFD8 QQ555,556EE were overexpressed, many filopodia-like processes extending from the main neurites were observed (A, B, and E). In contrast, neurons transfected with the TMFD8-G539P construct (D), with the TMFD8-C535G construct (F), or with the empty vector (C) showed only few filopodia-like processes. Quantification of the percent of process-containing tectal neurons (identified according to the criteria detailed under “Experimental Procedures”) after transfection with the TM-agrin mutation constructs is shown in G. All tested constructs that had process-inducing activity in HEK293 cells also induced the formation of processes in tectal neurons. Moreover, mutations that affected the process-inducing activity in HEK cells also resulted in a loss of the process formation activity in tectal neurons. Bars in G represent mean ± S.E. with n = 3. Scale bar = 20 μm.