Structure-function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function.
Secreted frizzled-related proteins (sFRPs) are glycoproteins that are recognized as Wnt antagonists. To identify the functional domains that are involved in Wnt antagonist function, several sFRP-1 mutants and sFRP-1/sFRP-2 chimeras were generated. These mutants were characterized in an optimized T-cell factor (TCF)-luciferase based assay in U2OS human osteosarcoma cells. Deletions of the sFRP-1 cysteine rich domain (CRD) lead to the complete loss of Wnt antagonist function. A region between amino acids 73-86 within the second loop of the CRD of sFRP-1 was necessary for the optimal Wnt inhibitory function. Within this region, a conserved tyrosine residue played a critical role, and its change to neutral or polar amino acids lead to decreased Wnt inhibitory activity. The sFRP-1/sFRP-2 chimeras with the netrin domain of sFRP-1 replaced by corresponding sFRP-2 sequences showed 40-70% loss of Wnt antagonist function. The sFRP-1/sFRP-2 chimera with the replacement of C-terminal 19 amino acids of sFRP-1 with 11 amino acids of sFRP-2 resulted in 70% loss of activity indicating that carboxyl-terminal region of sFRP-1 is important for its Wnt inhibitory activity. The structure-function analysis studies of sFRP-1 clearly demonstrate the interaction of several functional domains for its optimal Wnt antagonist function.
Bhat
Ramesh A
RA
Women's Health & Musculoskeletal Biology Division, Wyeth Research, Collegeville, Pennsylvania 19426, USA. Bhatr@Wyeth.com
Stauffer
Barbara
B
Komm
Barry S
BS
Bodine
Peter V N
PV
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000378:metabolism; D012516:Osteosarcoma / Q000473:pathology; D010957:Plasmids; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D051153:Wnt Proteins / Q000037:antagonists & inhibitors*; D000094742:Secreted Frizzled-Related Proteins
D036341:Intercellular Signaling Peptides and Proteins; D008156:Luciferases; D008565:Membrane Proteins; D050506:Mutant Chimeric Proteins; D014364:Tryptophan; D014443:Tyrosine; D051153:Wnt Proteins; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins