EVOLUTION OF THE PTH/PTHRP RECEPTOR AND ITS LIGANDS

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Parathyroid hormone (PTH), the most important regulator of mammalian calcium metabolism, is synthesized and secreted by the parathyroid glands; these are absent in fish and first appear in anurans before or during metamorphosis. PTH-related peptide (PTHrP), which has limited N- terminal amino acid sequence homology with PTH, is frequently responsible for the humoral hypercalcemia of malignancy (HHM) syndrome. Its normal physiological significance is poorly understood. Expression of PTHrP during early embryogenesis and in multiple fetal and adult tissues, however, suggests auto- or paracrine functions which are mediated through a receptor that is shared by PTHrP and PTH. The ligands and their receptor are thus likely to be of considerable importance for human development. Evolutionary studies as an approach to assess the function(s) of hormones, growth factors, and their effector pathways have proven useful for various endocrine/paracrine systems, and thus offer several unique opportunities to evaluate PTH and PTHrP: 1) the isolation of novel analogs for the structure/function analysis of ligand/receptor interaction and the definition of functionally important receptor domains, 2) novel, technically easier tools to study functions that are different or perhaps less obvious in mammalian systems, 3) powerful systems for genetic analysis, and 4) model systems to study the importance of PTHrP (and PTH) for human development. To gain insights into the physiological functions of PTHrP, to establish how PTH evolved into the major regulator of mammalian mineral ion metabolism, and to evaluate how both ligands diversified their actions through a common receptor, are the first goals of this proposal. We, therefore, propose in Aim I to study the evolutionary history of both ligands, their shared receptor, or possibly receptors that bind one or the other ligand preferentially or exclusively by isolating cDNAs encoding these proteins from chicken, Xenopus laevis, and fish, and subsequently from invertebrate species. the functional evaluation of PTH, PTHrP, and their common receptor from various species will, in Aim II, expand our knowledge of the structure/function relationship between ligands and receptors. Unique, species-specific features of either the ligands and/or the receptors will help to further define structurally and functionally important domains of the receptor. The resulting findings may allow the design of therapeutic agents for the treatment of hypercalcemia due to hyperparathyroidism or the HHM syndrome, and could have important implications for the understanding and the therapeutic approach towards osteoporosis. In Aim III, the above tools will be used to identify PTH-producing cells in pre- and post-metamorphosis anurans where parathyroid glands first appear, and in fish which have PTH, yet lack anatomically defined parathyroid glands. Furthermore, PTH and PTHrP will be evaluated in vivo in fish, target organs containing receptors for both ligands will be identified by Northern blot analysis, in situ hybridization and immunohistochemistry. The role of PTHrP (and PTH) in early embryogenesis will be evaluated in Xenopus laevis oocytes, blastomeres, and whole embryos by assessing mesoderm induction and patterning.