The recent cloning of porcine, human and murine calcitonin receptors (CTRs) by our laboratory and analysis of their structural features indicates that they belong to a new family of G protein-coupled receptors that includes the receptors for several members of the secretin/glucagon peptide family and parathyroid hormone/parathyroid hormone related peptide. The CTR cDNAs have provided us with experimental tools to define the molecular basis for the broad spectrum of biological and pharmacological effects of calcitonin (CT) and for determining the mechanisms underlying the observed cross-reactivity of CT with related ligands. The basis for the unique functional properties of the CTR, including the capacity to couple to multiple signal transduction pathways and to produce sustained activation of adenylate cyclase can also be explored. Initial studies indicate the presence of CTR isoforms and possibly subtypes with distinct functional properties. These findings provide the basis for Specific Aim 1 which is to clone and characterize the structural and functional properties of different CTR isoforms and related receptors. The observed structural and functional heterogeneity that we have detected among the CTR cDNAs that we have already cloned could proved the basis for tissue-specific regulation of responses to CT and related peptides.
Specific Aim 2 will use in situ hybridization with probes prepared from the CTR clones and immunohistochemistry with antibodies to the CTR, to establish the tissue distribution of these receptors. Because the CTR family of receptors exhibits structural features that are different from the other members of the G protein- coupled receptor super family, the structure/function relationships for these additional clone CTR cDNAs, to define the structural determinants underlying the unique functional properties of the CTR.
Specific Aim 3 proposes to use techniques of site-directed mutagenesis and construction of chimeric receptors to define the structural domains associated with ligand binding and coupling to G proteins associated with activation of specific signaling pathways. The development of refractoriness (""""""""escape"""""""") to the hypocalcemic effects of CT is a significant problem in the clinical and therapeutic use of CT. Insights into the mechanisms underlying this condition should lead to more effective use of CT in the treatment of disorders of skeletal remodeling. The goal of Specific Aim 4 will be to define the molecular and cellular basis of this state of refractoriness to CT and to establish its relationship to the processes of receptor desensitization and down-regulation.
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