PRIMARY STRUCTURE AND TISSUE DISTRIBUTION OF FRZB, A NOVEL PROTEIN RELATED TO DROSOPHILA FRIZZLED, SUGGEST A ROLE IN SKELETAL MORPHOGENESIS

Articular cartilage extracts were prepared to characterize protein fra ctions with in vivo chondrogenic activity (Chang, S., Hoang, B., Thoma s, J. T., Vukicevic, S., Luyten, F. P., Ryba, N. J. P., Kozak, C. A., Reddi, A. H., and Woos, M. (1994) J. Biol. Chem. 269, 28227-28234). Tr ypsin digestion of highly purified chondrogenic protein fractions allo wed the identification of several unique peptides by amino acid sequen cing. We discovered a novel cDNA encoding a deduced 36-kDa protein by using degenerate oligonucleotide primers derived from a 30-residue pep tide in reverse transcription polymerase chain reactions. Its N-termin al domain showed similar to 50%, amino acid identity to the correspond ing region of the Drosophila gene frizzled, which has been implicated in the specification of hair polarity during development. Hydropathy a nd structural analyses of the open reading frame revealed the presence of a signal peptide and a hydrophobic domain followed by multiple pot ential serine/threonine phosphorylation sites and a serine-rich C term inus. Cell fractionation studies of primary bovine articular chondrocy tes and transfected COS cells suggested that the protein is membrane-a ssociated. In situ hybridization and immunostaining of human embryonic sections demonstrated predominant expression surrounding the chondrif ying bone primordia and subsequently in the chondrocytes of the epiphy ses in a graded distribution that decreased toward the primary ossific ation center. Transcripts were present in the craniofacial structures but not in the vertebral bodies. Because it is expressed primarily in the cartilaginous cores of developing long bones during embryonic and fetal development (6-13 weeks) and is homologous to the polarity-deter mining gene frizzled, we believe that this gene, which we named frzb, is involved in morphogenesis of the mammalian skeleton.