OSTEOCALCIN RECEPTOR FUNCTION IN BONE RESORPTION

骨钙素(骨GLA蛋白)是含量最丰富的非胶原蛋白 在骨细胞外基质中，它被隔离在骨矿物质上 通过其2-3个残基的钙离子结合氨基酸-- 羧基谷氨酸(GLA)。成熟蛋白专一性生物合成骨钙素 成骨细胞受激素和有丝分裂调节因子的调节 成骨细胞和维生素D、K和C骨钙素是一种被广泛接受的 成骨细胞分化和骨形成的标志物。重要的是 骨钙素被破骨细胞和前体骨髓单核细胞识别 这种蛋白质显然是针对矿物质表面的细胞识别。 以及最终的再吸收。对这一分子细节的表征 生物学活性是理解骨吸收的关键 以及骨质疏松症的病理生理学。该项目寻求详细的 膜蛋白受体的分子和生理特性研究 人单核细胞系表达的骨钙素，特别是 1，25(OH)2维生素D3诱导HL-60早幼粒细胞模型。竞争性 与125I-骨钙素的结合和交联研究将确定受体 亲和力和分子量。亲和层析将用于 分离出足够用于胰酶的骨钙素受体 多肽分析和部分测序，为产生 受体抗体，为克隆提供了一条备用路线。一个主要目标 是利用哺乳动物克隆骨钙素受体基因 最近产生了许多其他多肽的表达克隆策略 受体cDNA。来自cDNA测序的信息将定义受体 类，并可预测信号转导机制。配基依赖 将直接在转基因灵长类动物COS中探索信号机制 高表达骨钙素受体的细胞和HL-60单核细胞。这个 骨钙素受体cdna在未来作为探针应该是非常有用的。 骨髓单核细胞破骨细胞受体基因的克隆及分离 分化途径。骨钙素的结构要求 人类单核细胞趋化和鸡的生物活性将被定义 体外激活破骨细胞，募集破骨细胞样巨细胞 体外培养鸡胚绒毛尿囊膜(CAM)和大鼠 体内骨颗粒吸收。竞争性多肽置换 结合的125I-骨钙素将用于定位骨钙素内部 测序单核细胞上骨钙素受体的表位(和 破骨细胞)，可能导致基于多肽的新疗法 减轻骨质疏松症和其他骨骼疾病的骨吸收。

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.