G-PROTEIN COUPLED RECEPTOR KINASES IN OSTEOGENESIS

描述（由申请人提供）：出现G蛋白偶联受体（GPCR） 有望成为促进骨生成的靶点。几种GPCR的激动剂 系统是新骨形成的有效诱导剂。与气相化学还原系统类似， 在其他组织中，GPCR在骨中的作用可能受到调节。这 调控可能涉及翻译后修饰的关键组成部分， 信号级联和/或与调节分子的相互作用。这些 机制，最广泛的研究是直接磷酸化受体 proteins. GPCR的磷酸化是GPCR快速衰减的原因。 在激动剂持续存在下发生的GPCR信号传导。这 脱敏可能限制GPCR激动剂对骨的有益作用。 因此，了解调节GPCR功能的机制可能 从而产生促进新骨形成的新策略。积累 有证据表明，GPCR的激动剂特异性脱敏在很大程度上是 通过受体丝氨酸和苏氨酸残基的直接磷酸化介导， 最近发现的受体特异性激酶（GPCR激酶或 GRKs）。激动剂刺激后，GRKs直接磷酸化GPCR， 减弱GPCR信号传导。在刺激骨生成的GPCR激动剂中， 甲状旁腺激素（PTH）已被最广泛地研究。当给予 无论是动物还是人，PTH都是骨的合成代谢物。在研究中，我们 假设GRKs在调节GPCR功能中起重要作用， 成骨细胞，并建议调查这些激酶的作用， 骨生成使用PTH受体作为模型系统。为了验证这个假设， 我们将采用三种相辅相成的办法。首先，我们将确定 GRK作用所需的PTH受体的结构组分， 已经用PTH受体cDNA转染。这种方法将允许 诱变研究，以及分离表位标记的PTH受体， 免疫沉淀和直接检查其磷酸化状态。 其次，我们将研究GRKs在调节PTH受体信号转导中的作用， 成骨细胞系这些研究将评估GRK的模式 在成骨细胞中的表达，以及调节GRK活性对 PTH受体反应性、细胞外基质积聚、细胞凋亡和 调节破骨细胞生成的成骨细胞因子的产生。最后， 我们将创建过表达GRKs或GRK抑制基因的转基因小鼠， 以骨特异性方式表达肽。在描述了它们的骨骼表型之后， 这些转基因动物将被用来研究不同的 PTH给药方案对骨形成的影响。这些研究应该提供新的， GRKs在调节骨生成中的作用的重要见解。 了解调节骨形成的生化机制可以 导致开发新的策略来治疗各种各样的骨 导致骨质疏松症的疾病。

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) appear promising as targets for promoting osteogenesis. Agonists of several GPCR systems are potent inducers of new bone formation. Similar to GPCR systems in other tissues, the effects of GPCRs in bone are likely to be regulated. This regulation may involve post-translational modifications of key components of the signaling cascade and/or interactions with regulatory molecules. Of these mechanisms, the most extensively studied is direct phosphorylation of receptor proteins. Phosphorylation of GPCRs is responsible for the rapid attenuation of GPCR signaling that occurs in the continuous presence of agonist. This desensitization may limit the beneficial effects of GPCR agonists on bone. Understanding the mechanisms that that regulate GPCR function might therefore lead to novel strategies for promoting new bone formation. Accumulating evidence suggests that agonist-specific desensitization of GPCRs is largely mediated by direct phosphorylation of receptor serine and threonine residues by a recently discovered family of receptor specific kinases (GPCR kinases or GRKs). Following agonist stimulation, GRKs directly phosphorylate GPCRs and attenuate GPCR signaling. Of the GPCR agonists that stimulate osteogenesis, parathyroid hormone (PTH) has been the most extensively studied. When given to either animals or humans, PTH is anabolic for bone. In the proposed studies, we hypothesize that GRKs play important roles in modulating GPCR function in osteoblasts and propose to investigate the role of these kinases in osteogenesis using the PTH receptor as a model system. To test this hypothesis, we will use three complementary approaches. First, we will determine the structural components of PTH receptor required for GRK actions using cells that have been transfected with the PTH receptor cDNA. This approach will permit mutagenesis studies, as well as isolation of epitope-tagged PTH receptors by immunoprecipitation and direct examination of their phosphorylation state. Second, we will study the role of GRKs in modulating PTH receptor signaling in osteoblast cell lines. These studies will evaluate the patterns of GRK expression in osteoblasts, as well as the effect of modulating GRK activity on PTH receptor responsiveness, extracellular matrix accumulation, apoptosis and production of osteoblastic factors which modulate osteoclastogenesis. Lastly, we will create transgenic mice that overexpress either GRKs, or GRK inhibitory peptides in a bone-specific manner. After characterizing their bone phenotype, these transgenic animals will be used to investigate the effect of different PTH dosing regimens on bone formation. These studies should provide new, important insights into the role of GRKs in regulating osteogenesis. Understanding the biochemical mechanisms that regulate hone formation could lead to the development of novel strategies for treating a wide variety of bone disorders that cause osteoporosis.