Regulation of TGFβ superfamily signaling by two separable domains of glypican LON-2 in C. elegans

Abstract

Regulated intercellular signaling is critical for the normal development and maintenance of multicellular organisms. Glypicans have been shown to regulate signaling by TGFβs, hedgehogs and Wnts, in several cellular contexts. Glypicans comprise a conserved family of heparan sulfated, glycosylphosphatidylinositol (GPI)-linked extracellular proteins. The structural complexity of glypicans may underlie their functional complexity. In a recent study(31), we built on previous findings that one of the two C. elegans glypicans, LON-2, specifically inhibits signaling by the TGFβ superfamily member DBL-1. We tested the functional requirements of LON-2 protein core components and post-translational modifications for LON-2 activity. We provide the first evidence that two parts of a glypican can independently regulate TGFβ superfamily signaling in vivo: the N-terminal furin protease product and a C-terminal region containing heparan sulfate attachment sites. Furthermore, we show a protein-protein interaction motif is crucial for LON-2 activity in the N-terminal protein core, suggesting that LON-2 acts by serving as a scaffold for DBL-1 and an RGD-binding protein. In addition, we demonstrate specificity of glypican function by showing C. elegans GPN-1 does not functionally substitute for LON-2. This work reveals a molecular foundation for understanding the complexity and specificity of glypican function.

Keywords: LON-2; RGD-binding protein; TGFβ signaling; body size; glycosylphosphatidylinositol anchor; glypican; heparan sulfate proteoglycan.

Figure 1. Model of LON-2 parts that regulate DBL-1/TGF-β superfamily signaling. (A) The full-length LON-2 inhibits DBL-1 signaling and prevents a long body length phenotype. Full-length LON-2 (shown in A) remains functional if it lacks any one of the sequences containing RGD, all heparan sulfate attachment sites or the GPI anchor site. (B) The cylindrical N-terminal core protein, LON-2(1–368), requires the RGD motif for function. (C) The unstructured C-terminal 86 amino acid region, LON-2(423–508), requires both heparan sulfate attachment sites and a GPI anchor sequence to inhibit DBL-1 signaling. Arrows indicate parts required for function of the LON-2 variants shown in B and C. This diagram is based on tertiary structure predictions from the crystal structures of Dally-like and human GPC1.^,