SCUBE2 [signal peptide, CUB domain, EGF (epidermal growth factor)-like protein 2] belongs to an evolutionarily conserved SCUBE protein family, which possesses domain organization characteristic of an N-terminal signal peptide sequence followed by nine EGF-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C-terminus. Despite several genetic analyses suggesting that the zebrafish orthologue of the mammalian SCUBE2 gene participates in HH (Hedgehog) signalling, the complete full-length cDNA and biochemical function for mammalian SCUBE2 on HH signalling remains uninvestigated. In the present study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signalling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cysteine-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with SHH (Sonic Hedgehog) and SHH receptor PTCH1 (Patched-1), and enhance the SHH signalling activity within the cholesterolrich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel positive component of the HH signal, acting upstream of ligand binding at the plasma membrane. Thus human SCUBE2 could play important roles in HH-related biology and pathology, such as during organ development and tumour progression.
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