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Identification and Characterization of the Zebrafish and Fugu Genes Encoding Tuberoinfundibular Peptide 39

Department of Biological Sciences (M.R.P., B.P., D.A.R.), Illinois State University, Normal, Illinois 61790; Endocrine Unit (R.C.G., Y.G., H.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; Institute of Neuroscience (Y.-L.Y., J.H.P.), University of Oregon, Eugene, Oregon 97403; and Novartis Institutes for BioMedical Research (M.R.J.), Bone Metabolism Research, CH-4002 Basel, Switzerland

Address all correspondence and requests for reprints to: David A. Rubin, Department of Biological Sciences, Illinois State University, Normal, Illinois 61790. E-mail: adavid{at}ilstu.edu.

Although the PTH type 2 receptor (PTH2R) has been isolated from mammals and zebrafish, only its mammalian agonist, tuberoinfundibular peptide 39 (TIP39), has been characterized thus far. To determine whether zebrafish TIP39 (zTIP39) functions similarly with the zebrafish PTHR (zPTH2R) and human PTH2Rs and to determine its tissue-specific expression, fugu (Takifugu rubripes) and zebrafish (Danio rerio) genomic databases were screened with human TIP39 (hTIP39) sequences. A single TIP39 gene was identified for each fish species, which showed significant homology to mammalian TIP39. Using standard molecular techniques, we isolated cDNA sequences encoding zTIP39. The fugu TIP39 precursor was encoded by a gene comprising at least three exons. It contained a hydrophobic signal sequence and a predicted prosequence with a dibasic cleavage site, similar to that found in mammalian TIP39 ligands. Phylogenetic analyses suggested that TIP39 forms the basal group from which PTH and PTHrP have been derived. Functionally, subtle differences in potency could be discerned between hTIP39 and zTIP39. The human PTH2R and zPTH2R were stimulated slightly better by both hTIP39 and zTIP39, whereas zTIP39 had a higher potency at a previously isolated zPTH2R splice variant. Whole-mount in situ hybridization of zebrafish revealed strong zTIP39 expression in the region of the hypothalamus and in the heart of 24- and 48-h-old embryos. Similarly, zPTH2R expression was highly expressed throughout the brain of 48- and 72-h-old embryos. Because the mammalian PTH2R was also most abundantly expressed in these tissues, the TIP39-PTH2R system may serve conserved physiological roles in mammals and fishes.

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