The life span of an organism is regulated by multiple factors, including endocrine mediators, which affect many relevant processes at both the cellular and organismal levels. Insulin-like growth factor 1 (IGF-1) pathway has been recognized as one of the most critical endocrine factor impacting aging: low levels of serum IGF-1 are negatively correlated with longevity. Recent work in our lab has characterized a novel control mechanism of IGF-1 production and secretion - an obligatory interaction with the chaperone Glucose Regulated Protein 94 (GRP94). In the absence of active GRP94, IGF-1 folding intermediates fail to mature, are not secreted and thus are not available for signaling. Furthermore, the biosynthesis of IGF-1 is proportional to the activity of GRP94 and mutants of GRP94 with partial activity support IGF-1 production to varying degrees. The hypothesis of this project is that functional polymorphisms of the human GRP94 gene exist, affect the level of IGF-1 production and thus affect human aging. This hypothesis will be tested by: 1. Sequencing for rare GRP94 coding variants in order to relate sequence variation of GRP94 to IGF-1 levels in the human population. Two cohorts in the Longevity Consortium will be used for this Aim. 2. Testing genome wide association between either rare or common SNPs in the GRP94 gene and the levels of circulating IGF-1. 3. Testing the functionality of the variants discovered in Aim 1 via a cell-based assay in which the ability of cells to survive under stress is proportional to the production of IGF under the control of active GRP94. 4. Further investigation will describe the role of posttranslational modifications (Ser/Thr phosphorylations) of GRP94 that occur in the lumen of ER and relate to its activity and substrate specificity (i.e. secretion of IGF-1). To date, the feasibility of each of these aims has been demonstrated. Two human GRP94 mutations that affect activity have been described. These and additional mutants will demonstrate for the first time a causative relationship between an ER protein, IGF-1 signaling and regulation of human longevity. Phosphorylation of GRP94 has been described;clarification of its functional significance and description of the first ER resident kinase will construe a new level of communication and regulation of signaling between cell environment, cytoplasm signaling and ER. The proposed Mentored Research Career Development Award will enable me to mature and transition to an independent scientific career. This goal will be achieved by: a) acquiring new knowledge in ER physiology, human genomic studies and statistical analysis;b) expanding laboratory skills to include embryonic stem cell culture, genetic manipulations, gene sequencing, fluorescent microscopy, image analysis and mass spectrometry;c) improvement of teaching skills;d) refinement of scientific writing skills;e) interactions with different types of collaborators (through the Health ABC and LonGenity Studies);and f) exposure to the scientific community through attendance and presentations in national meetings.
This work will test the hypothesis that sequence variations of the human GRP94 gene affect the levels of insulin-like growth factors produced by tissues and thereby affect one of the determining factors in aging. Genotyping and sequencing will be used to discover GRP94 variants, which will then be tested in a novel cellular assay. In the later stages of the proposal physiological functions of posttranslational modifications of GRP94 will be investigated. This project will expand my scientific skills and will help me transition into an independent academic career.