Our initial application proposed to study the mechanism of action and regulation of the calcitonin receptor (CTR) gene. Although the ultimate goal was to use this information to gain insights into the control of osteoclast differentiation and function, we have broadened the scope our studies to include investigation of the regulation and mechanism of action of the CTR in organs and tissues not directly involved in mineral ion homeostasis. We have established that the human CTR gene encodes multiple CTR isoforms that are functionally distinct and are expressed in a tissue- and cell-specific fashion. These observations have provided insights into the diversity of the biological activities of calcitonin (CT). In the competing renewal, we will continue to focus attention on the osteoclast CTR, but will also investigate the receptor in other CT-responsive tissues.
The Aims are as follow: 1. Utilize the reverse transcriptase/polymerase chain reaction (RT-PCR) and cloning techniques to complete the sequencing and characterization of the structural properties of the human CTR isoforms. The corresponding genomic structure will be identified and the tissue and cell-specific distribution of the isoforms established using in situ hybridization, Northern blot analysis and RNase protection. 2. Complete the characterization of cells in specific bone lesions, e.g. Paget's disease of bone and other granulomatous diseases and tumors in which the phenotype of the giant cells and their relationship to osteoclasts is not well established. This will help to define the phenotypic relationship between bone resorbing cells in physiological and pathological remodeling and identify the structural features of the osteoclast-associated CTR . 3. Because the CTR family of receptors exhibit structural features that are different from other members of the G protein-coupled receptor superfamily, the structure/function relationships for these receptors are of particular interest. We propose to utilize the different CTR cDNAs presently available, as well as additional CTR isoforms that we have recently identified, to define the unique signaling properties and binding kinetics of the human CTR isoforms. 4. In the final Specific Aim, we propose to clone and characterize the human CTR gene and to define the specific and potentially unique regulatory sequences responsible for expression of the CTR in osteclasts and other CTR-expressing cells and tissues. Constructs of the CTR 5'-region containing the putative promoter sequences of the CTR gene will be linked to reporter gene(s) and screened for the capacity to induce CTR expression using transfection in human mononuclear cell lines or other cell types that constitutively express the CTR (e.g. kidney). This will define the structural basis of CTR gene regulation and permit identification of potentially unique factors that regulate the CTR.
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