OSTEOCALCIN RECEPTOR FUNCTION IN BONE RESORPTION

骨钙素（骨Gla蛋白）是最丰富的非胶原蛋白 在骨细胞外基质中，它被骨矿物质隔离 通过其2-3个Ca 2+结合氨基酸γ- 羧基谷氨酸（Gla）。 骨钙素的专一性生物合成 成骨细胞受激素和促有丝分裂调节剂的调节， 成骨细胞和维生素D K 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， 骨钙蛋白被破骨细胞和前体骨髓单核细胞识别， 这种蛋白质显然以矿物质表面为细胞识别目标 最终再吸收。 分子细节的表征 生物活性对于理解骨吸收至关重要 和骨质疏松症的病理生理学。 该项目寻求详细 膜受体的分子和生理特性 人单核细胞系表达的骨钙素，特别是 用1，25（OH）2维生素D3诱导HL-60早幼粒细胞模型。 竞争 用125 I-骨钙素的结合和交联研究将确定受体 亲和力和分子量。 亲和色谱法将用于 分离出足够量的骨钙素受体， 肽分析和部分测序，为产生 受体抗体，并为克隆提供备用途径。 一个主要目标 是利用哺乳动物的骨钙素受体cDNA， 表达克隆策略，最近已经产生了许多其他肽 受体cDNA。 来自cDNA测序的信息将确定受体 类，并可能预测信号转导机制。 配体依赖性 将在转染的灵长类COS中直接探索信号传导机制 骨钙素受体过表达的细胞和HL-60单核细胞。 的 骨钙素受体cDNA作为探针在将来会非常有用 受体基因的克隆及骨髓单核细胞-破骨细胞的分离 分化途径 骨钙素的结构要求 将针对人单核细胞趋化性和鸡单核细胞趋化性定义生物活性。 体外破骨细胞活化，破骨样巨细胞募集 在鸡胚绒毛尿囊膜（CAM）体外培养中， 体内骨颗粒吸收。 竞争性肽置换 结合的125 I-骨钙素将被用于定位在骨钙素内 对单核细胞上骨钙素受体的表位进行测序（和 破骨细胞），可能导致新的，基于肽的治疗， 减弱骨质疏松症和其它骨骼疾病中的骨吸收。

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.