Isolation and characterization of a secreted, cell-surface glycoprotein SCUBE2 from humans.
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 cholesterol-rich 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.
Tsai
Ming-Tzu
MT
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Cheng
Chien-Jui
CJ
Lin
Yuh-Charn
YC
Chen
Chun-Chuan
CC
Wu
Ann-Ru
AR
Wu
Min-Tzu
MT
Hsu
Cheng-Chin
CC
Yang
Ruey-Bing
RB
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D051242:Caveolin 1 / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D006031:Glycosylation; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016366:Open Reading Frames / Q000235:genetics; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D015398:Signal Transduction; D029961:Zebrafish Proteins / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D051242:Caveolin 1; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D053823:Hedgehog Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; C000606240:Ptch1 protein, mouse; D011956:Receptors, Cell Surface; C469999:SCUBE2 protein, human; C509239:SHH protein, human; C500835:Scube2 protein, zebrafish; D029961:Zebrafish Proteins; C491715:ptch1 protein, zebrafish