Mechanisms of Lrrk1 Regulation of Osteoclast Function

描述（由申请人提供）：Leucine rich repeat kinase 1 （Lrrk1）属于ROCO家族蛋白，包含3个锚蛋白重复序列、7个富含亮氨酸重复序列、Roc的gtpase样结构域（复杂蛋白的Ras）、COR （Roc的c -末端）结构域和GTP结合到Roc结构域调控的丝氨酸/苏氨酸激酶结构域。Lrrk2包含ROCO家族蛋白的大部分结构域和一个额外的Lrrk2特异性重复序列，但缺乏ankryrin重复序列。Lrrk1和Lrrk2在多种组织中普遍表达。我们最近发现，Lrrk1基因破坏的小鼠表现出严重的多核细胞功能障碍引起的骨质疏松表型，而缺乏Lrrk2基因的小鼠没有表现出明显的骨骼表型，这表明Lrrk1在骨细胞中具有独特的结构和功能，Lrrk2不能弥补Lrrk1的缺失。对Lrrk1在破骨细胞中的信号通路知之甚少。我们在这项资助中的重点是确定Lrrk1 i破骨细胞的结构和功能，并确定潜在的Lrrk1激酶靶点和抑制剂。为此，我们在本研究中提出两个假设：1)Lrrk1通过其激酶激活调节破骨细胞功能；2）

DESCRIPTION (provided by applicant): Leucine rich repeat kinase 1 (Lrrk1) belongs to ROCO family proteins and contains three ankyrin repeat, seven leucine-rich repeat, a GTPase-like domain of Roc (Ras of complex proteins), COR (C-terminal of Roc) domain, and a serine/threonine kinase domain that is regulated by GTP binding to the Roc domain. Lrrk2 contains most domains of ROCO family protein and an extra Lrrk2 specific repeat, but lacks ankryrin repeat. Both Lrrk1 and Lrrk2 are ubiquitously expressed in multiple tissues. We recently found that mice with disruption of Lrrk1 gene displayed a severe phenotype of osteopetrosis caused by dysfunction of multinucleated cells while mice lacking Lrrk2 gene failed to show obvious skeletal phenotypes, indicating that Lrrk1 has a unique structure and function in bone cells that Lrrk2 cannot compensate for loss of Lrrk1. Little is known on the signaling pathways of Lrrk1 in osteoclasts. Our focus in this grant is to determine the structure and function of Lrrk1 i osteoclasts and identify potential Lrrk1 kinase targets and inhibitors. To this end, we propose two hypotheses in this study: 1) Lrrk1 regulates osteoclast functions via its kinase activation; 2) Lrrk1 phosphorylates serine/threonine residues of its specific substrate(s). To test the hypothesis 1, experiments are designed to examine the structure and function of full length Lrrk1 and truncated Lrrk1 on bone resorptive activity of mature osteoclasts on bone slices by in vitro pit assay. Homology models of human Lrrk1 kinase domain are used to dock small molecule inhibitors and the inhibition of drug-like compounds will be tested in vitro kinase and pi formation assays. To test the hypothesis 2, experiments are designed to enrich phosphor-peptides from wild type and Lrrk1 deficient osteoclasts via immunoaffinity purification by using phosphor-serine/phosphor-threonine motif antibodies, and identify Lrrk1 initial targets by liquid chromatography and tandem mass spectrometry (LC- MS/MS). The results of this application will advance our understanding of the molecular mechanisms of Lrrk1 signaling in regulating osteoclast function and bone resorption, and provide new therapeutic strategies for treatment and prevention of bone diseases such as osteoporosis.