David A Rubin, PhD - US grants

The PTH/PTHrP receptor (PPR) mediates the biological actions of two peptide hormones, PTH and PTH-related peptide (PTHrP). PTH is the major regulator of mammalian mineral ion homeostasis. PTHrP and the common PPR are both crucial for normal development, since the disruption of either gene in mice results in lethal phenotypes with widespread abnormalities of endochondral bone formation. These "knockout" studies indicated furthermore that the common PPR is essential for mediating the functions of PTH and PTHrP, and that no other receptor can serve as a substitute. However, a novel, PTH-specific receptor (PTH2R) with significant amino acid sequence homology to the PPR was isolated from mammals, but its biological importance is not yet known. Interestingly, cDNAs encoding fish PTH2R homologs were recently isolated also from catfish and zebrafish cDNA libraries. If these novel receptors are activated only by PTH, as the mammalian PTH2R homolog, these findings suggest that a PTH-like molecule exists also in vertebrate species that do not have parathyroid glands and that PTH expression occurs in species lacking distinct parathyroid glands. In the current proposal, I will therefore study the coevolution and function of two closely related G protein-coupled PTH-/PTHrP-receptors and their two distinct ligands. The resulting findings are expected to provide evidence for possible function(s) of both ligands and both receptors in nonmammalian vertebrates, and may provide further clues for biological role(s) in mammals that are distinct from those required for the control of mineral ion homeostasis.

DESCRIPTION (provided by applicant): A G-protein coupled PTH/PTHrP type-2 receptor (PTH2R) from teleosts and mammals shows significant homology to the vertebrate PTHfPTHrP type-1 receptor (PTH1R). In an effort to isolate the cognate ligand of the PTH2R, a peptide of 39 amino acid residues was purified from the bovine hypothalamus (tuberoinfundibular peptide of 39 residues, bTIP39), which selectively and potently stimulates the mammalian and teleost PTH2R. It therefore appears that TIP39 is the endogenous ligand of the vertebrate PTH2R. Recently, genomic DNA and cDNA sequences for human and murine TIP39 were isolated, the encoded ligands characterized in vitro, and their tissue specific expression identified in many diverse adult neuroendocrine tissues. In addition, the VTH2R has been shown to have a spatial and temporal expression during early development in the kidney, brain, and testis. To date, only several zebrafish putative PTH2R splice variants have been identified; unknown are the identification of genomic regulatory elements for the human and zebrafish PTH2Rs, as well as TIP39. The identification of promoters for TIP39 and the PTH2R may clarify their tissue specific expression and provide significant information regarding the physiological ramifications of this newly discovered endocrine system. To test the hypothesis that the TIP39-PTH2R system has a regulated expression in diverse endocrine and neuronal tissues, this proposal describes the molecular cloning of the gene and transcript for zebrafish TIP39, identification of the corresponding promoters (Specific Aim 1), and functional characterization of zebrafish TIP39 (Specific Aim 2). In addition, this proposal will clone the zebrafish PTH2R gene to determine the regulatory elements, which control the spatial and temporal expression of the PTH2R and the putative splice variants (Specific Aim 3). The novelty and innovation of using zebrafish as an animal model provides a powerful model system to study the role of TIP39 and the PTH2R in development of several neuro-endocrine systems, which may provide further clues for their biological roles in mammals. Therefore, this work will initiate a long-term molecular endocrine and developmental investigation of the role of the TIP39-PTH2R system in early vertebrate development. Furthermore, future studies may be initiated in characterizing the molecular mechanisms regulating the TIP39 and PTH2R gene expression.

DESCRIPTION (provided by applicant): Tuberoinfundibular peptide 39 (TIP39) and Parathyroid Hormone (PTH) type-2 receptor (PTH2R) constitute a new endocrine system with conserved neuronal mRNA expression in zebrafish and mouse brain. The functions associated with the murine TIP39-PTH2R system include nociception and regulation of hypothalamic (GnRH) and pituitary hormones (ACTH, GH, and LH). Zebrafish studies have shown that TIP39 and the PTH2R are expressed early in brain development in a spatial and temporal relationship with sonic hedgehog (Shh). Furthermore, embryos which have TIR39 expression inhibited by morpholino are not viable and show severely impaired brain development. Thus, the zebrafish TIP39-PTH2R studies suggest that this system serves a crucial role in neuroendocrine development and perhaps other organs such as pancreas and heart where TIR39 and the PTH2R are expressed. Therefore, the zebrafish, Danio rerio, provides a powerful model system to study TIR39-PTH2R-regulated mechanisms during development of the central nervous system (CNS). Hence, molecular analyses are proposed to better elucidate the effects of altered zebrafish PTH2R and TIR39 transcript expression during early brain development. Three aims will be pursued, 1) Evaluation of PTH2R and TIP39 MO-injected embryos during early brain development by examining the expression of Shh, GH, ACTH, LH, and GnRH. 2) Determine the relationship between TIP39 and Shh during brain development by examining the locale of cells expressing TIP39 in embryos which have a deficiency for Shh or Shh signaling (sonic you and slow-muscle-omitted). 3) Evaluation of PTH2R and TIP39 overexpression in the brain and hypothalamus (respectively) during early brain development by examining the expression of the marker genes described above. Because the PTH2R has not been analyzed by gene knockout in any organism, these experiments will provide novel insights into the role of this new TIP39-PTH2R system in normal and perturbed embryos. This work is part of a long-term molecular developmental-genetic investigation on the mechanistic role(s) of TIP39-PTH2R system during CNS, and other organ, development.