Primary hyperparathyroidism (PHPT) is characterized by inappropriate parathyroid hormone (PTH) secretion by neoplastic parathyroid cells lacking normal responsiveness to calcium-dependent negative feedback. This disruption in the homeostatic relationship between calcium and circulating PTH levels is operationally described as an altered calcium-PTH setpoint. Current dogma stipulates that the altered calcium sensing observed in PHPT patients results from reduced expression of the G-protein coupled calcium sensing receptor (CASR) in parathyroid tumor cells. However, several lines of evidence indicate that reduced CASR abundance is not the only determinant of abnormal calcium responsiveness in PHPT. First, we and others have found that reduced expression of CASR is not uniformly observed among parathyroid tumors or within individual tumors. Immunohistochemical analysis of parathyroid adenomas reveals heterogeneity in CASR expression with no correlation between CASR abundance and plasma PTH levels or gland size. Moreover, the relative calcium responsiveness of dispersed parathyroid cells in vitro varies widely and does not correlate with CASR expression. Second, cluster analysis of transcriptional microarray data generated in our laboratory reveals at least three distinct subtypes of parathyroid tumors distinguished in part by differential levels of CASR-related gene expression. Finally, we have found that subgroups of parathyroid adenomas express high levels of the GAP protein Regulator of G protein signaling 5 (RGS5), which we show is capable of inhibiting CASR biochemical signaling in vitro. In vivo, mice lacking RGS5 express reduced levels of circulating PTH, consistent with the predicted consequences of unopposed CASR activity. Collectively, these data suggest that perturbations in CASR signaling in PHPT are not solely determined by relative CASR abundance. In this proposal, we will test the hypothesis that molecular events including but not limited to downregulation of CASR expression are responsible for abnormal calcium sensing by parathyroid tumors. Towards this overall objective, we will pursue three specific aims: (1) we will develop a molecular categorization paradigm for parathyroid tumors based upon the relative expression patterns of CASR and RGS5; (2) we will investigate the mechanisms of RGS5 overexpression in parathyroid tumors and will determine the effect of RGS5 on calcium signaling in dispersed parathyroid cells; and (3) we will assess the impact of RGS5 gene ablation on the hyperparathyroidism phenotype expressed by two established murine models of PHPT and determine whether tissue-specific overexpression of RGS5 in the murine parathyroid gland can induce a PHPT phenotype. These experiments will establish a mechanistic framework for understanding the role of RGS5 in the pathogenesis of PHPT and will provide a foundation for developing more effective targeted approaches to addressing the many metabolic disorders associated with dysfunctional calcium homeostasis. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to identify the causes of abnormal calcium regulation in patients who have a relatively common condition known as hyperparathyroidism (HPT). Currently, this hormonal imbalance disease is treated surgically by removal of the abnormal parathyroid gland. By analyzing the proteins whose function are lost or broken in parathyroid gland tumors, we will be able to determine how the parathyroid gland can maintain normal regulation of calcium content in the body. Furthermore, we will be able to use these results to better understand the nature of parathyroid tumors and how they arise from normal tissue. Finally, by learning more about why parathyroid tumors don't sense calcium properly, we will be able to identify promising new approaches for treating patient with HPT using medications rather than surgery.