Primary Structure and Tissue Distribution of FRZB, a Novel Protein Related to Drosophila Frizzled, Suggest a Role in Skeletal Morphogenesis

doi:10.1074/jbc.271.42.26131

Primary Structure and Tissue Distribution of FRZB, a Novel Protein Related to Drosophila Frizzled, Suggest a Role in Skeletal Morphogenesis*

From the ^‡ Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892,
^¶ Laboratory of Developmental Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, and
^∥ Department of Anatomy, Medical School of Zagreb, 41000 Zagreb, Croatia

^″ To whom correspondence should be addressed:
Bone Research Branch, Bldg. 10, Rm. 1N108, NIDR, National Institutes of Health, Bethesda, MD 20892-1188.
Tel. and Fax: 301-402-3502; E-mail: luyten{at}yoda.nidr.nih.gov

Abstract

Articular cartilage extracts were prepared to characterize protein fractions with in vivo chondrogenic activity (Chang, S., Hoang, B., Thomas, J. T., Vukicevic, S., Luyten, F. P., Ryba, N. J. P., Kozak, C. A., Reddi, A. H., and Moos, M. (1994) J. Biol. Chem. 269, 28227-28234). Trypsin digestion of highly purified chondrogenic protein fractions allowed the identification of several unique peptides by amino acid sequencing. We discovered a novel cDNA encoding a deduced 36-kDa protein by using degenerate oligonucleotide primers derived from a 30-residue peptide in reverse transcription polymerase chain reactions. Its N-terminal domain showed ~50% amino acid identity to the corresponding region of the Drosophila gene frizzled, which has been implicated in the specification of hair polarity during development. Hydropathy and structural analyses of the open reading frame revealed the presence of a signal peptide and a hydrophobic domain followed by multiple potential serine/threonine phosphorylation sites and a serine-rich C terminus. Cell fractionation studies of primary bovine articular chondrocytes and transfected COS cells suggested that the protein is membrane-associated. In situ hybridization and immunostaining of human embryonic sections demonstrated predominant expression surrounding the chondrifying bone primordia and subsequently in the chondrocytes of the epiphyses in a graded distribution that decreased toward the primary ossification 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-determining gene frizzled, we believe that this gene, which we named frzb, is involved in morphogenesis of the mammalian skeleton.

Footnotes

↵^§ Supported by the Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U24163[GenBank] for human FRZB and U24164[GenBank] for bovine Frzb.
↵¹ The abbreviations used are:

fz

frizzled

PCR

polymerase chain reaction

RT-PCR

reverse transcription-PCR

kb

kilobase pair(s)

PBS

phosphate-buffered saline

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
↵² frzb is pronounced frizbee; frz from frizzled motif and b from bone development.
↵³ J. T. Thomas and F. P. Luyten, unpublished observations.
- Received February 26, 1996.
- Revision received July 15, 1996.