Abstract
We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that, although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.
Highlights
We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2
Our previous studies had shown that extracellular epimorphin, when presented in combination with a growth factor such as EGF, is sufficient to direct mammary epithelial branching morphogenesis; we had further localized the morphogenic activity of epimorphin to a domain of the molecule contained within the first 187 amino acids, a fragment that we designated as H12 [1, 3]
To determine whether this property is unique to epimorphin among the syntaxin family of proteins, we used a three-dimensional collagen branching assay to compare the morphogenic capacity of this fragment of EPM with a fragment of syntaxin-1a that lacks the transmembrane domain (Syn1a)
Summary
C/EBP, CCAAT/enhancer-binding protein-; MMP3, matrix metalloproteinase-3; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; LAP, liver-activating protein; LIP, liver-inhibitory protein; EPM, epimorphin; Habc, N-terminal autonomously folded 3-helix bundle domain; EGF, epidermal growth factor. We use structural and functional information about syntaxins to create an epimorphin homology model and to deduce the site of a key epimorphin ligand binding motif; we demonstrate the specificity of the interactions formed by this motif using site-directed mutagenesis to create an active morphogen from the functionally inactive syntaxin-1a. These results identify precisely the minimal structural motif of epimorphin essential for its function as a morphogen and bring us closer to understanding its mode of interaction with critical physiological ligands
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