Osteocalcin (bone Gla protein) is the most abundant non-collagenous protein in bone extracellular matrix, where it is sequestered on bone mineral through its 2-3 residues of the Ca2+ binding amino acid gamma- carboxyglutamate (Gla). Exclusive biosynthesis of osteocalcin by mature osteoblastic cell is regulated by hormonal and mitogenic modulators of osteoblasts and vitamins D, K, and C. Osteocalcin is a widely accepted marker for osteoblastic differentiation and bone formation. Importantly, osteocalcin is recognized by osteoclasts and precursor myelomonocytes and this protein apparently targets mineral surfaces for cellular recognition and eventual resorption. Characterization of the molecular details of this biological activity is critical for the understanding of bone resorption and the pathophysiology of osteoporosis. This project seeks detailed molecular and physiological characterization of the membrane receptor for osteocalcin as expressed by human monocyte cell lines, particularly the model HL-60 promyelocyte induced with 1,25(OH)2 vitamin D3. Competitive binding and crosslinking studies with 125I-osteocalcin will define receptor affinity and molecular weight. Affinity chromatography will be used to isolate quantities of the osteocalcin receptor sufficient for tryptic peptide analysis and partial sequencing, paving the way for generation of receptor antibodies and providing a backup route for cloning. A major goal is to clone the osteocalcin receptor cDNA utilizing the mammalian expression cloning strategy which has recently yielded many other peptide receptor cDNAs. Information from cDNA sequencing will define the receptor class and may predict signal transduction mechanism. The ligand-dependent signalling mechanism will be explored directly in transfected primate COS cells overexpressing the osteocalcin receptor and in HL-60 monocytes. The osteocalcin receptor cDNA should be extremely useful as a probe in future cloning of the receptor gene and dissection of the myelomonocyte-osteoclast differentiation pathway. Structural requirements for osteocalcin bioactivity will be defined for human monocyte chemotaxis and chicken osteoclast activation in vitro, recruitment of osteoclast-like giant cells in chick embryo chorioallantoic membrane (CAM) cultures in vitro, and rat bone particle resorption in vivo. Competitive peptide displacement of bound 125I-osteocalcin will be used to locate within the osteocalcin sequence the epitope for the osteocalcin receptor on monocytes (and osteoclasts), possibly leading to new, peptide-based therapies for attenuating bone resorption in osteoporosis and other skeletal disorders.
