This application is in response to NOT-OD-09-058, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." It is a competitive revision request for grant RO1 DE 007444, "Bone matrix and bone resorption," under which we are investigating the osteoclast cell biology of a protein we discovered called plekhm1. Evidence indicates that plekhm1 is required for normal trafficking of vesicles, secretion, and bone resorption by osteoclasts. Mutations that truncate the plekhm1 protein cause osteopetrosis and inactive osteoclasts in a rat model and in human patients. In contrast, a point mutation, R714C, was recently discovered by our collaborators in a human patient, which appears to cause excessive osteoclast activity. This leads to increased secretory activity by osteoclasts and an osteopenic skeleton. The current project is examining binding partners that interact with plekhm1 to control vesicle movement and secretion in osteoclasts, as well as its regulation by bioactive lipids. This proposal would add a new specific aim to our ongoing project. We hypothesize that the plekhm1-R714C mutation is a dominant, gain-of-function mutation that leads to excessive bone loss. We will test this hypothesis under the new specific aim by generating a mouse model in which the endogenous, wild type plekhm1 is replaced by gene knock-in with plekhm1-R714C. The skeletal phenotype of the mice will be evaluated by DEXA, pQCT and microCT. Serum TRAP levels will also be measured to determine the extent to which the mouse line phenocopies the human patient who is heterozygous for the point mutation. TEM and immune-EM studies will examine mutant osteoclasts. Cells derived from the knock-in mice will be used to investigate the alterations in osteoclast biology and plekhm1 binding and regulation caused by the point mutation. A new post-doctoral fellow will be hired to carry out this project, which will last for 2 years. We are also requesting funds to purchase a fluorescence microscope with which we can both perform routine screening for tagged protein expression in cell cultures and perform deconvolution confocal imaging experiments to screen candidate proteins for co-localization with plekhm1, as part of a complex required for normal vesicle trafficking, secretion, and bone resorption by osteoclasts. PUBLIC HEALTH RELEVANCE: Project narrative: Widespread bone disorders, including osteoporosis, arthritis, periodontal bone disease, joint replacement loosening, and tumor metastasis to bone, all have in common bone loss that exceeds bone formation. Bone loss is carried out by cells called osteoclasts, and the more deeply we understand how osteoclasts work, the more opportunities will arise for potential therapeutics to control their activity. This proposal aims to investigate a new gene we discovered which causes bone disease in animal models and in human patients. Deeper understanding of this gene and how it controls the bone resorbing activity of osteoclasts will give a better knowledge of bone biology in general, and potentially will lead to new ways to treat these all-too-common bone disorders.