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Endocrinology Vol. 143, No. 3 1047-1057
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Identification and Characterization of the Murine and Human Gene Encoding the Tuberoinfundibular Peptide of 39 Residues

Markus R. John1, Maya Arai, David A. Rubin, Kenneth B. Jonsson and Harald Jüppner

Endocrine Unit (M.R.J., M.A., K.B.J., H.J.), Department of Medicine and MassGeneral Hospital for Children (H.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and Department of Biological Sciences (D.A.R.), Illinois State University, Normal, Illinois 61790

Address all correspondence and requests for reprints to: Harald Jüppner, M.D., Endocrine Unit, Wellman 5, 50 Blossom Street, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: . jueppner{at}helix.mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
By screening public databases, we identified human and mouse genomic DNA clones that encode the tuberoinfundibular peptide of 39 residues (TIP39). The TIP39 precursor is encoded by at least three exons; a noncoding exon U1, exon 1 encoding residues -61 (initiator methionine) to -19 of the leader sequence, and exon 2 encoding residues -18 to -1 and residues +1 to +39. Secreted human TIP39 is identical to the previously isolated bovine TIP39, whereas mouse TIP39 differs by four amino acids. Phylogenetic analyses suggested that TIP39, PTH, and PTHrP may have evolved from a common ancestor. Synthetic human and mouse TIP39 showed indistinguishable potencies [EC50: 0.54 (human) vs. 0.74 nM (mouse)] at the human PTH2-receptor stably expressed in LLCPK1 cells; furthermore, TIP-(9–39) was an inhibitor of cAMP accumulation stimulated by either [Tyr34]PTH(1–34)amide or human/bovine TIP39. In the mouse, an approximately 4.5-kb mRNA encoding TIP39 was identified by Northern blot analysis in testis and, less abundantly, in liver and kidney, whereas other tissues revealed additional smaller transcripts. In situ hybridizations revealed TIP39 expression in seminiferous tubuli and several brain regions, including nucleus ruber, nucleus centralis pontis, and nucleus subparafascicularis thalami. Because PTH2 receptor expression was previously shown to be highest in brain, pancreas, and testis, our findings are consistent with the notion that TIP39 is a neuropeptide which may also have a role in spermatogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
THE TUBEROINFUNDIBULAR PEPTIDE of 39 residues (TIP39) was recently purified from bovine hypothalamic extracts and the complete amino acid sequence of the mature peptide was obtained by microsequence analysis (1). TIP39 appears to be distantly related to PTH and PTHrP because nine amino acid residues, some of which have been shown to be functionally important in both latter peptides (2), are either conserved or identical among all three peptides. Although PTH was shown to efficiently activate the human type 2 PTH receptor (PTH2 receptor) (2, 3, 4), this peptide was later shown to interact only weakly with rat and zebrafish homologs of this receptor (5, 6). Furthermore, PTHrP activated none of the known PTH2 receptor homologs, although this peptide was shown to bind, albeit with reduced affinity, to this subfamily of receptors (2, 4, 7). Because this finding suggested that PTH and PTHrP are not the primary ligand for the PTH2 receptor, a search for a novel agonist at this receptor was begun. Initial studies revealed that bovine hypothalamic extracts, which failed to activate the PTH/PTHrP receptor, efficiently stimulated cAMP accumulation in cells expressing the rat or the human PTH2 receptor (8). Subsequent efforts led to the isolation and definition of the primary structure of a novel peptide, referred to as TIP39, from bovine hypothalamus and the synthetic peptide was shown to efficiently activate human, rat, and zebrafish PTH2 receptors, but not PTH/PTHrP receptors from several different species (1, 9, 10). TIP39 rather than PTH (or PTHrP) thus appears to be the primary ligand for the PTH2 receptor. However, native TIP39 and some of its amino-terminally truncated analogs were shown to bind to the PTH/PTHrP receptor and to act as competitive antagonists of PTH- or PTHrP-stimulated cAMP accumulation (10, 11). Three distinct peptides, PTH, PTHrP, and TIP39 that share only limited amino acid sequence homology thus interact with the PTH/PTHrP receptor.

Little is thus far known about the physiologic role(s) of the TIP39-PTH2 receptor system. The PTH2 receptor is expressed in somatostatin-expressing hypothalamic periventricular neurons, which suggested a possible role in the regulation of GH release (1). It is also expressed in the spinal cord, within the superficial layers of the dorsal horn, indicating that TIP39 may be involved in pain perception (1). Furthermore, TIP39 may be identical or related to a hypothalamic substance that stimulates renin release in the juxta-glomerular apparatus of the kidney (12), where the PTH2 receptor is expressed (13), and may thus have a role in blood pressure regulation.

We now report the identification of genomic DNA sequences encoding human and murine TIP39, the organization of both mammalian genes, and a partial functional characterization of the mature peptides from both species. Furthermore, we provide an initial assessment of the tissue distribution of mouse TIP39 mRNA, and of the phylogenetic relationship between TIP39, PTH, and PTHrP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Identification of genomic clones encoding human and mouse TIP39, chromosomal location of their genes, and predictions regarding the cleavage of the precursor peptides
Partial genomic nucleotide sequence encoding TIP39 was obtained by searching the high throughput genomic sequence draft sequences of the National Center for Biotechnology Information (NCBI) with the bovine TIP39 amino acid sequence (TBLASTN search). Nucleotide sequence alignment of human and murine genomic DNA encoding TIP39 was performed using the NCBI Blast 2 sequences server with default parameters (http://www.ncbi.nlm.nih.gov/gorf/bl2.html), the GCG Wisconsin Package, or MacVector 7.0 software (both from Genetics Computer Group, Madison, WI). Information regarding the chromosomal localization of human TIP39 was obtained by searching the database of the Genome Sequencing Center at Washington University School of Medicine (St. Louis, MO) with nucleotide sequence information from different BAC clones (http://genome.wustl.edu/gsc/human/Mapping/index.shtml). Additional sequence information was obtained by using the Human Genome Browser of the University of Santa Cruz (Santa Cruz, CA) (http://genome.cse.ucsc.edu) and the Ensembl Genome Server of the EMBL European Bioinformatics Institute (http://www.ensembl.org). Putative cleavage sites within the TIP39 precursor were predicted using the neural network approach of SignalP V2.0b2 of the Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark (http://www.cbs.dtu.dk/services/SignalP-2.0/) (14, 15). This program was also used to predict cleavage sites for human PTH and PTHrP, which allowed verification of the computer program through previously published experimental data (16, 17).

Peptides
Human TIP-(1–39) and TIP-(9–39), mouse TIP-(1–39), [Tyr34] human PTH-(1–34)amide [PTH-(1–34)] were synthesized by the Biopolymer Core Facility at Massachusetts General Hospital (Boston, MA) using Fmoc chemistry on Applied Biosystems (Foster City, CA) synthesizers (model 430A or 431A). All peptides were purified to homogeneity by reversed-phase chromatography, and amino acid sequences were confirmed by analysis of amino acid composition and amino acid sequence, and by mass spectroscopy.

Cell culture and stimulation of cAMP accumulation
DMEM, Trypsin/EDTA, penicillin G/streptomycin, and horse serum were from Life Technologies, Inc. (Gaithersburg, MD). LLCPK1 cells expressing the human PTH2 receptor, clone hPR2–20 (approximately 0.8 x 106 copies/cell), were kindly provided by F. R. Bringhurst, Endocrine Unit, Massachusetts General Hospital (Boston, MA). Cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, in a humidified atmosphere containing 95% air and 5% CO2, as previously described (2, 18). After seeding into 48-well plates, medium was replaced every other day. Upon confluence, cells were used for stimulating cAMP accumulation. Agonist-dependent stimulation of cAMP accumulation was performed at room temperature for 45 min, and the subsequent measurement of cAMP by RIA was performed as previously described (2, 19). Data were analyzed and graphically displayed using the Prism 3.0 software package (GraphPad Software, Inc., San Diego, CA).

Rapid amplification of cDNA ends (RACE)
To identify the 5' end of the cDNA encoding human TIP39, 5' RACE was performed by using a Marathon-Ready cDNA kit to amplify cDNAs from human hypothalamus (CLONTECH Laboratories, Inc., Palo Alto, CA). The initial PCR was performed using the provided AP1 primer and a primer specific for human TIP39 (hTIPr5: 5'-AGCAGCTTGTGCATGTACGAG-3'). A 50-µl reaction consisted of 5 µl hypothalamic cDNA, 1 µl AP1 primer, 1 µl hTIPr5 primer (100 pmol), 1 µl (2U) polymerase (GC-rich polymerase, Roche Molecular Biochemicals, Indianapolis, IN), 1 µl deoxynucleoside triphosphates (dNTPs) (10 mM each, Roche Molecular Biochemicals), 10 µl PCR buffer, 5 µl GC-rich solution, and 31 µl H2O. The following optimized reaction profile was carried out using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany): initial denaturation at 98 C for 1 min and at 95 C for 2 min; subsequent program: denaturation at 95 C for 30 sec, annealing at 69 C for 30 sec, polymerization at 72 C for 2 min; after the first cycle, the annealing temperature was decreased by 1 C for each of the following 4 cycles. Subsequently, 35 cycles were performed with denaturation at 95 C for 2 min, annealing at 63 C for 30 sec, and polymerization at 72 C for 2 min; final extension at 72 C for 7 min. Five microliters of the diluted product (1:50 in H2O) was reamplified in a nested PCR using 1 µl AP2 primer, 1 µl hTIPr4 primer (5'-TTGTGCATGTACGAGTTCAGC-3'; 100 pmol), and the same reaction profile as before. This reaction was electrophoresed through a 2% agarose gel and stained with ethidium bromide. For molecular cloning, 4 µl of the final PCR product were ligated into pCR 2.1-TOPO (Invitrogen) for transformation of TOP10 cells. Plasmid DNA was prepared by standard techniques and DNA sequence analysis was performed at the Massachusetts General Hospital core sequencing facility.

RT-PCR
Approximately 1 µg of poly-A+ RNA from murine brain (Ambion, Inc., Austin, TX) was reverse transcribed using a primer specific for murine TIP39 (mTIP2rev: 5'-GTCCAGTAGCAACAGCTTCTGC-3'; 100 pmol) and the Omniscript II reverse transcriptase kit (QIAGEN, Hilden, Germany) at 42 C for 1 h (final reaction volume: 20 µl). One tenth of the reaction was used for an initial PCR, which consisted of 2 µl (100 ng) reverse-transcribed template DNA, 1 µl dNTPs (10 mM each, Roche Molecular Biochemicals), 1 µl (100 pmol) mTIPCR2-f6 forward primer (5'-TCTCTATTTTTATCCCTCTGAC-3'; 100 pmol), 1 µl (100 pmol) mTIP2rev primer, 5 µl PCR-Buffer (QIAGEN), 10 µl Q-solution (QIAGEN), 0.5 µl HotStar Taq polymerase (QIAGEN), and 29 µl H2O. The reaction profile was: initial denaturation at 95 C for 15 min, then 35 cycles with denaturation at 94.5 C for 30 sec, annealing at 65 C for 45 sec, polymerization at 72 C for 30 sec; final extension at 72 C for 10 min. A nested PCR using 2 µl of the initial reaction product was performed using forward primer mTIPCR2-f5 (5'-CTCTGACACACCCCTTGTGTC-3'; 100 pmol) and reverse primer mTIP2rev following the same reaction profile. Four microliters of the final reaction product were ligated into pCR 2.1-TOPO (Invitrogen) for transformation of TOP10 cells.

Preparation of a cDNA encoding portions of murine TIP39
A 103-bp genomic DNA fragment encoding portions of murine TIP39 was PCR-amplified using the following reaction profile: 1 µl (200 ng) mouse genomic DNA, 1 µl mTIP5for primer (5'-CTAGCTGACGACGCGGCCTTTCG-3'; 100 pmol), 1 µl mTIP2rev primer (100 pmol), 1 µl dNTPs (10 mM, Roche Molecular Biochemicals), 5 µl PCR buffer, 1 µl dimethylsulfoxide, 0.5 µl Pfu-Turbo polymerase (Stratagene, La Jolla, CA), and 39.5 µl H2O; initial denaturation at 98 C for 1 min and at 95 C for 3 min, denaturation at 95 C for 45 sec, annealing at 69.5 C for 1 min, polymerization at 72 C for 30 sec; after the first cycle, the annealing temperature was decreased by 1 C for each of the following 4 cycles; subsequently, 35 cycles with denaturation at 95 C for 45 sec, annealing at 64.5 C for 1 min, polymerization at 72 C for 30 sec; final extension at 72 C for 5 min. The reaction was electrophoresed through a 4% agarose gel and stained with ethidium bromide. Forty microliters of the reaction were purified using the QIAquick PCR purification kit (QIAGEN) and eluted with 30 µl H2O. Four microliters of the eluate was ligated into pCR 4Blunt-TOPO (Invitrogen) for transformation of TOP10 cells. Nucleotide sequence and orientation of the insert was confirmed by nucleotide sequence analysis using a M13 reverse primer (Massachusetts General Hospital core sequencing facility).

Northern blot analysis
A mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc.) with 2 µg poly(A)+-RNA from eight different tissues was probed with the cDNA encoding mouse TIP39 (nucleotides 1 to 472; AY048587). After excision from the vector using EcoRI (New England Biolabs, Inc., Beverly, MA) and purification, approximately 50 ng of the cDNA encoding TIP39 were random-labeled with 32P-dCTP using the Prime-a-Gene labeling system (Promega Corp., Madison, WI). The blot was prehybridized in 5 ml of ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.) (72 C for 3 h) and hybridized with 5 ml of hybridization solution containing the labeled probe (1.5 h at 72 C). Four 15-min washes with 2x SSC (0.3 M NaCl, 0.03 M trisodium citrate, adjust pH to 7.0 with 1 M HCl), 0.1% SDS were performed at room temperature; subsequently two washes, 20 min each, were performed with 0.1x SSC, 0.1% SDS at 50 C and the blot was exposed for 3 d using Kodak X-OMAT AR films (Kodak, Rochester, NY).

In situ hybridization
Fresh frozen tissue sections were prepared from 10- to 12-wk-old adult mice; 10-µm tissue sections were mounted on Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA) and stored at -80 C until hybridization. The hybridization procedure was performed as described (20) using complementary 35S-labeled riboprobes (cRNAs). The antisense probe was transcribed from the pCR 4Blunt plasmid comprising 103 bp of murine TIP39 (see above) using the T3 polymerase; the sense probe, which served as negative control, was transcribed from the same plasmid using the T7 polymerase. Slides were covered with Kodak NTB-2 emulsion and exposed for 2–4 wk, before developing and staining with hematoxylin and eosin (H&E) (20). Electronic images were obtained with both bright and dark field optics using a Nikon (Melville, NY) photomicroscope.

Phylogenetic analysis
To further explore whether TIP39 is related to PTH and PTHrP, phylogenetic analyses were performed using all currently available species of these three peptides. With the exception of equine PTH and bovine TIP39 for which precursor sequences were not available, complete amino acid sequences that included the signal peptides were used for alignment by CLUSTAL W (21). These aligned amino acid sequences were subsequently entered into MacClade 4.0 (22) with manual adjustments as described (23). These data were analyzed for either distance (Neighbor-Joining) or parsimony (Maximum Parsimony) using PAUP version 4.0b8 (24). For each analysis, 10,000 bootstrap and jackknife replicates were carried out on the entire set, in which the human gastrointestinal-inhibitory peptide (GIP) was used as the outgroup, while secretin (human, pig, and mouse), VIP (human, mouse, and chicken), and all known homologs of PTH, PTHrP, and TIP39 formed the ingroups. Analysis was performed also on a modified data set lacking the signal peptides.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Identification of BAC clones encoding human and mouse TIP39
TBLASTN homology searches of the GenBank Nucleotide Sequence database (high throughput genomic sequence, draft sequences) were performed using the entire amino acid sequence of bovine TIP39. We identified two unordered BAC clones, one human clone (accession no. AC068670) encoding a peptide that was 100% identical to secreted bovine TIP39, and one mouse clone (accession no. AC073763) encoding a peptide that showed four amino acid differences when compared with the human/bovine TIP39 amino acid sequence (Fig. 1AGo). No additional genomic sequences encoding peptides with significant amino acid sequence homology to human/bovine TIP39 were identified.



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  		Figure 1. A, Schematic representation of the known portions of the human (upper panel) and the mouse (lower panel) TIP39 gene. The names of the different exons are indicated; the sizes of exons (normal letters) and introns (italic letters) are given in base pairs; the approximate positions of the different PCR primers are shown (see Materials and Methods); note that the positions of the universal AP1 and AP2 primers that were used for 5' RACE are arbitrary. B, Splice donor/acceptor sites in the human and mouse gene are shown; exonic nucleotides are shown in capital letters; intronic nucleotides in lowercase letters; splice site consensus nucleotides are in bold; the initiator ATG in exon 1 is underlined.

 
Searching the database of the Genome Sequencing Center at Washington University School of Medicine (St. Louis, MO) (http://genome.wustl.edu/gsc/human/Mapping/index.shtml) with clone AC068670 revealed that the genetic locus for human TIP39 resides on chromosome 19q13.33. This clone is flanked toward the centromer by the fully sequenced and assembled BAC clone AC024079.2, and toward the telomer by the fully sequenced and assembled clone AC011495.6; it partially overlaps furthermore with the finished clones AC011450.4, AC008891.7, AC010524.6 and AC010643.5, and with the unordered clones AC068786.11, and AC010619.5. Within this genetic region are several microsatellite markers, including D19S987 and D19S669E, which are located centromeric and telomeric of TIP39, respectively. A total of 70 single nucleotide polymorphisms in BAC clone AC068670 are currently available from database single nucleotide polymorphism (dbSNP; www.ncbi.nlm. nih.gov/SNP), which may also be helpful for genetic linkage studies. The mouse genomic region corresponding to human chromosome 19q13.33 is located on mouse chromosome 7.

Gene structure and cDNA encoding TIP39
To determine the intron-exon structure of the murine and human TIP39 gene, we first aligned genomic DNA fragments derived from human BAC clone AC068670 and mouse BAC clone AC073763. The alignment of two 2150-bp fragments from these clones revealed four regions of particularly high nucleotide identity between mouse and human genomic DNA, whereas the intervening sequences showed less nucleotide sequence identity (Fig. 1AGo). The first region, referred to as CR2, contained 55 bp and showed 96% nucleotide sequence identity between mouse and human genomic DNA. A second conserved region, located 34 bp downstream of CR2, contained 145 bp that showed 81% nucleotide sequence identity and was subsequently found to comprise portions of exon U1. Two additional regions comprising 207 bp and 180 bp showed 82% and 81% nucleotide sequence identity; these regions were subsequently found to contain exons 1 and 2 of mouse and human TIP39.

For both mammalian species, the most 3' region (exon 2) contained an open reading frame (ORF) encoding the entire secreted TIP39, followed by a consensus sequence for polyadenylation that is located in both mammalian genes 21 nucleotides downstream of the termination codon. Fifty-four nucleotides further upstream of the sequences encoding the mature TIP39s, potential splice sites were identified in both species and the nucleotide sequence identity decreased thereafter. The next region with higher nucleotide sequence homology (exon 1) contained in both species an ORF encoding a putative initiator methionine (residue -61) and a stretch of thirty hydrophobic amino acids (residues -51 to -22) that could serve as leader sequences; for mouse and human genomic DNA these ORFs were flanked by nucleotide sequences possibly representing splice sites (Figs. 1BGo and 2Go). Based on these findings, mouse and human cDNAs were both predicted to encode TIP39 precursors comprising 100 amino acids. However, it remains uncertain whether additional exons exist that could give rise to alternatively spliced mRNAs that are either larger or smaller in size, and encode peptides that differ in size (see below).



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  		Figure 2. Nucleotide sequence of the human TIP39 gene. Nucleotides found in the mature mRNA are capitalized, nucleotides in flanking intervening DNA sequences are in lowercase. Because of uncertainty about the start site of transcription and the exact length of exon U1, the first nucleotide of the coding region is designated nucleotide +1. Splice donor and acceptor sites are underlined; a putative polyadenylation signal is shown in bold underlined lowercase letters. Partial exon U1 sequence information (deduced from mouse TIP39) is in dark gray. Coding nucleotides are shaded in light gray. The amino acid sequence of the human precursor TIP39 is indicated below nucleotides. The secreted peptide sequence is boxed.

 
To confirm these predictions regarding the size of the mammalian cDNAs, to identify possibly untranslated exons, and to investigate whether several differently spliced mRNAs are derived from the two mammalian TIP39 genes, we performed 5' RACE using commercially available adult human hypothalamic cDNA and RT-PCR using murine brain poly-A+ RNA. Only a single product of approximately 310 bp encoding human TIP39 was obtained by nested PCR amplification of human hypothalamic cDNA (using primers "AP2" and "hTIPr4", see Fig. 1AGo). Nucleotide sequence analysis of this PCR product confirmed that human TIP39 is indeed encoded by two exons (exons 1 and 2), i.e. the 569 bp of intronic nucleotide sequence predicted based on the comparison between murine and human TIP39 had been excised. However, because the PCR product derived from the human hypothalamic cDNA library contained only 7 novel bp at the 5' end (which furthermore represented a consensus splice sequences), no information regarding noncoding sequences upstream of the putative initiator AUG was available. Additional PCRs using the human hypothalamic cDNA and primers located further upstream failed to provide additional 5' untranslated nucleotide sequence.

We therefore reverse transcribed total mRNA from mouse brain with primer mTIP2rev and performed nested PCRs using this primer and additional mouse-specific forward primers located in those genomic regions that showed the highest nucleotide sequence homology when comparing mouse and human genomic DNA (CR2 and exon U1) (see Fig. 1AGo). When using primers mTIPCR2-f5 and mTIP2rev, three nested PCR products were obtained, cloned, and sequenced. The largest PCR product of approximately 900 bp corresponded to the genomic DNA sequence and was therefore most likely derived from contaminating TIP39 genomic DNA or from pre-mRNA. A PCR product of approximately 650 bp lacked the intronic sequence between exons 1 and 2, while the intervening sequence between exons 1 and U1 was present. This suggested that the latter product was most likely derived from partially processed pre-mRNA. The smallest PCR product of approximately 560 bp comprised a nucleotide sequence extending from exon U1 to exon 2, which did not appear to contain intronic DNA sequences. Furthermore, no additional conserved splice sites were present in the 5' region of this cDNA sequence, indicating that the mRNA from which this PCR product was derived had been completely processed. At least for the mouse TIP39 gene, these findings thus confirmed not only the predicted intron between exons 1 and 2, but also the intron between exons U1 and 1 that had been predicted based on the nucleotide sequence alignment of mouse and human genomic DNA clones (see Fig. 1Go, A and B, and Fig. 2Go). No additional differently spliced mRNAs and/or additional 5' untranslated exons were detected.

The TIP39 sequence around the putative initiator ATG was only partially in the context of the usual consensus sequence for the initiation of translation (CGGUGAUGG in mouse and human; deviation from the perfect Kozak consensus is italicized) (see Fig. 1BGo). However, because a guanine or an adenine at position -3 and a guanine at position +4 appear to be the most important nucleotides flanking the initiator AUG (25), translation of TIP39 mRNA should readily occur. An in-frame termination codon was identified 18 nucleotides upstream of the putative AUG in the mouse mRNA, but not in the human gene (see Fig. 2Go).

The cDNAs (GenBank accession nos.: AY048588 for human TIP39; AY048587 for mouse TIP39) encoding human and mouse TIP39 showed 80% identity across the entire coding sequences, whereas the deduced amino acid sequence was 97%/90% similar/identical for the two mature peptides. Human and mouse TIP39 precursors are both predicted to comprise 100 amino acids with an overall amino acid similarity/identity of 84%/78% (Fig. 3AGo); the predicted presequences alone (61 amino acids) showed less homology (77%/72% similarity/identity). The cDNAs encoding both TIP39 precursor were found to be particularly rich in guanine and cytosine (GC-content: 74.3% and 69.7%, respectively) compared with a human genome-wide average of 41% (26). The intervening sequences between exons U1 and 1, and between exons 1 and 2 had GC-contents of 66.6% and 59.6%, and 58.7% and 65.9%, respectively (human vs. mouse). No expressed sequence tags derived from the human or mouse TIP39 gene were identified when searching the NCBI GenBank databases.



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  		Figure 3. A, Amino acid sequence alignment for the human and murine TIP39 precursors. Residues that are identical (dark shade) or similar (light shade) in human and mouse TIP39 are shown; the black bar depicts the secreted peptide with the first residue denoted as "+1". B, Kyte/Doolittle hydrophobicity plot of the deduced human TIP39 precursor (upper panel) and mouse TIP39 precursor (lower panel) amino acid sequence. The thick black bar depicts the secreted peptide; the position of the first residue is denoted as "+1". The ordinate indicates relative hydrophobicity, with more positive values corresponding to increased hydrophobicity.

 
Comparison of the genes encoding TIP39, hPTH, and hPTHrP
As outlined above, human and mouse TIP39 shared a high degree of structural homology. Furthermore, both genes share organizational features with the genes encoding PTH and PTHrP. Like the PTH gene (27), TIP39 consists of at least three exons, including one exon comprising the 5' UTR. In contrast, the PTHrP gene is more complex in that it comprises several additional coding and noncoding exons, which give rise to several different mRNA transcripts (16, 28). While TIP39 is most likely synthesized as a longer precursor which contains an additional 61 amino acids at the amino terminus, the precursors of PTH and PTHrP comprise shorter preprosequences (i.e. 31 and 36 amino acids, respectively). For all three genes, the message derived from the exon encoding the 5' UTR is spliced onto the first coding exon. For PTH and PTHrP, this exon encodes all but two amino acids of the prepro-sequence, whereas the first coding exon of TIP39 encodes only about two thirds of the much longer partially hydrophobic leader sequence (i.e. amino acid residues -61 to -19) (Fig. 3BGo). The remaining portion of the precursor sequence is encoded by exon 2, i.e. the equivalent of the exons encoding mature PTH and PTHrP (Fig. 4Go).



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  		Figure 4. Comparison of the gene structure for human TIP39, human PTH, and human PTHrP. Boxed areas are exons and their names are shown underneath (because the start of exon U1 of the TIP39 gene is unknown, the box is open on the left side), white boxes denote presequences, black boxes denote prosequences (for TIP39 presumed), gray stippled boxes denote the mature sequences; noncoding regions are shown as striped boxes. The small striped boxes preceding the white boxes denote untranslated exonic sequences (4 bp for TIP39; 5 bp for PTH; 22 bp for PTHrP). The positions of the initiator methionine based on the secreted peptide are noted above the graphs; the positions where prosequences are interrupted by an intron are noted above the graph. +1 denotes the relative position of the beginning of the secreted peptide.

 
PTH, PTHrP, and TIP39 must undergo posttranslational processing to yield biologically active peptides. While processing of the PTH and PTHrP precursors was previously explored (for review see Refs. 29 and 30), there are no experimental data yet exploring the generation of mature TIP39 from its precursor. Using the neural network algorithm provided by the SignalP World Wide Web server (14, 15), signal peptide cleavage sites were predicted for human and mouse TIP39 between amino acid residues -32 and -31 (human residues: VRT-AS; mouse residues: TGP-AS). Using the same algorithm, the established processing sites for PTH and PTHrP were correctly predicted, making it plausible that the cleavage sites predicted for the two mammalian TIP39 molecules are indeed correct. It is currently unknown whether the TIP39 precursor contains, similar to PTH and PTHrP, a presequence and a prosequence. However, the amino acid sequence preceding the cleavage site between the putative prohormone and the mature peptide contains two basic residues in both mammalian TIP39 species. These residues are typically found at the end of prosequences [31, including PTH and PTHrP (16, 27)], and residues -31 to -1 could thus represent the prosequence of TIP39.

Phylogenetic analysis of TIP39, PTH, and PTHrP
Amino acid sequence comparison of the receptors for secretin, calcitonin, PTH-PTHrP, and several other peptides of intermediate in length revealed a close phylogenetic relationship, thus establishing the class B family of G protein-coupled receptors (32, 33). It is furthermore well established that PTH and PTHrP evolved through an ancient gene-duplication event from a common precursor (30). Because TIP39 shares some amino acid sequence homology with PTH and PTHrP, and because all three peptides interact not only with the PTH/PTHrP receptor, but also with the PTH2 receptor, we assessed whether the genes encoding these peptides could be derived from a common ancestor. Amino acid sequence alignment of all known PTH and PTHrP molecules, as well as murine, bovine, and human TIP39 were aligned and analyzed by distance and parsimony methods. Several secretin and vasoactive intestinal polypeptide (VIP) species were included in vasoactive intestinal polypeptide (VIP) the analysis because these peptides share within the amino acid sequences of their signal and secreted peptides several parsimony-informative characters with PTH, PTHrP, and TIP39. Phylogenetic inference furthermore strongly suggested that human GIP can be used as an appropriate outgroup (34, 35).

Although the terminal branches differed depending on whether Maximum Parsimony or Neighbor-Joining analyses were performed, all trees showed the same topology of groups, i.e. distinct groups for PTH, PTHrP, TIP39, and secretin. The highest bootstrap and jackknife values were obtained by Neighbor-Joining analysis when including the full-length precursor proteins, which included the signal peptides (Fig. 5Go); present theories indicate that nodes with bootstrap values above 95% are considered strongly supportive of a close phylogenetic relationship (36, 37). Although minor differences in the phylogenetic relationship within the clades for PTH and PTHrP could not be resolved due to the limited amount of characters available for analysis, significant phylogenetic differences in the relationships between PTH, PTHrP, and TIP39 groups became apparent. Consistent with the previously postulated gene duplication event (30), our results indicate that PTH and PTHrP are closely related sister groups. Furthermore, even though the precursor sequence was not available for bovine TIP39, which may alter the overall significance values, TIP39 grouped strongly as the sister group to the PTH-PTHrP superfamily, implying that all three groups of ligands are derived from a common ancestor. However, the isolation of peptides with similarities to PTH, PTHrP, and TIP39 from lower vertebrate species will be required to confirm this hypothesis.



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  		Figure 5. Phylogenetic analysis indicating the evolutionary relationship among precursor proteins of the TIP39, PTH, PTHrP, and secretin families of peptides. A Neighbor-Joining phylogenetic analysis using distance as the criteria is shown above (tree length = 740, consistency index excluding uninformative residues = 0.876, with 165 parsimony-informative characters). The bootstrap/jackknife values from 10,000 replicates indicate support of a given node where 95% is considered to be significant (36 37 ). A Maximum Parsimony analysis using parsimony as the criteria generated a similar phylogenetic relationship between PTH, PTHrP, TIP39, secretin, and GIP (tree length = 746, consistency index excluding uninformative residues = 0.846, with 165 parsimony-informative characters). In addition to the Neighbor-Joining and Maximum Parsimony phylogenetic analyses, Quartet puzzling using Maximum Parsimony criteria and Star-decomposition (tree length = 739, consistency index excluding uninformative characters = 0.855, with 165 parsimony-informative characters) (35 36 ) support the hypothesis that PTH and PTHrP are sister groups, and that TIP39 is the sister group to this clade. PTH, PTHrP, and TIP39 thus form a superfamily, whereas secretin and VIP (not shown) appears to be a sister group to this larger superfamily (accession nos.: PTH (cat, AF309967; chick, M36522; cow, J00024; dog, U15662; horse, AF134233; human, NM_000315; macaque, AF130257; mouse, NM_020623; pig, X05722; and rat, NM_017044); PTHrP (chick, X52131; dog, U15593; cow, P58073; human, J03580; mouse, M60056; rabbit, AF219973; rat, NM_012636; sheep, AF327654; fugu, AJ249391; sparus, AF197904); VIP (chick, U09350; mouse AK018599; human XM_004381); secretin [mouse, X73580; pig, M31496; human, XM_012014; and human GIP (NM_004123)].

 
Characterization of the TIP39 gene product
To determine whether mouse and human TIP39 (which is identical to bovine TIP39) activate the human PTH2 receptor with similar efficiency, we synthesized both peptides and assessed agonist-induced cAMP accumulation in LLCPK1 cells stably expressing this receptor. Both peptides showed equal potency and efficacy at this receptor (EC50 for human TIP39: 0.54 nM; EC50 for mouse TIP39: 0.74 nM; maximum cAMP accumulation: 136.5 ± 4.9 pmol/well and 133.7 ± 3.9 pmol/well, respectively) (Fig. 6AGo). Analogs of TIP39 were recently shown to be potent inhibitors of PTH-(1–34) action at the PTH/PTHrP receptor (10, 11, 38). We therefore tested whether TIP-(9–39) can antagonize the actions of PTH-(1–34) and human TIP39 at the PTH2 receptor. The actions of either agonist, at concentrations that induced half-maximal cAMP accumulation in cells expressing the PTH2 receptor were inhibited by TIP-(9–39). The activity of 1 nM PTH-(1–34) was inhibited by TIP-(9–39) with an IC50 of 1.1 x 10-7 M, whereas approximately 14-fold higher concentrations of the antagonist were required to antagonize cAMP accumulation stimulated by 1 nM human TIP39 (IC50: 4 x 10-6 M) (Fig. 6BGo). The efficacy of TIP-(9–39) as an antagonist at the PTH2 receptor thus appears to be similar to that of [Nle8,18, D-Trp12, Tyr34]-bPTH-(7–34)NH2 and [Leu11, D-Trp12]hPTHrP-(7–34)NH2 at the PTH/PTHrP receptor (39), which may be sufficient to help exploring the biological roles of TIP39.



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  		Figure 6. A, Ligand-stimulated cAMP accumulation in hPR2–20 LLCPK1 cells stably expressing the recombinant human PTH2 receptor. Cells were stimulated with increasing concentrations of human TIP-(1–39) ({bullet}) or mouse TIP-(1–39) ({blacktriangleup}). Data are expressed as picomoles per well and represent the results (mean ± SEM) of two independent experiments; basal cAMP accumulation was 0.23 pmol/well. B, Inhibition of agonist stimulated cAMP accumulation in hPR2–20 LLCPK1 cells. Cells were stimulated with approximately half-maximal concentrations of human TIP-(1–39) ({bullet}) or PTH-(1–34) ({blacksquare}) in the absence or presence of increasing concentrations of TIP-(9–39). Data are expressed as percentage of half-maximal cAMP accumulation and represent the results (mean ± SEM) of two independent experiments.

 
TIP39 expression
Transcripts encoding the human PTH2 receptor were initially detected by Northern blot analysis in poly-A+ RNA from brain, pancreas, testis, and placenta (3). In situ hybridization studies subsequently revealed mouse mRNA transcripts in glomeruli, somatostatin synthesizing D cells of the pancreatic islets, and numerous areas of the brain, including the preoptic area of the periventricular nucleus, the diagonal band of Broca, the amygdala, the arcuate nucleus, ventromedial nucleus and dorsal paraventricular nucleus among other areas (13, 40, 41).

Northern analysis using a blot with poly-A+ RNA (2 µg/lane) from multiple mouse tissues revealed a prominent message of approximately 4.5 kb in testis, which was also observed, albeit at much lower intensity, in liver, kidney, and possibly heart (Fig. 7Go). Poly-A+ RNA from testis furthermore revealed two larger transcripts, while poly-A+ RNA from liver showed evidence for very weakly hybridizing transcripts of about 1.5 kb, and poly-A+ RNA from brain showed transcripts of 1.0 kb and possibly 0.7 kb. Thus, with the exception of testis, TIP39 does not seem to be abundantly expressed. The presence of the large TIP39 mRNA transcripts suggests that its gene may comprise more exons than currently known. It is also plausible that TIP39 comprises a longer 3' noncoding region than suggested by the presence of a consensus polyadenylation signal just downstream of the termination codon, or that the hybridizing mRNA in testis represents incompletely processed pre-mRNA.



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  		Figure 7. Northern blot analysis of poly-A+ RNA derived from several different mouse tissues using a cDNA probe encoding mouse TIP39 (nucleotides 1–472; AY048587; see Materials and Methods). Note that poly-A+ RNA from testis showed three hybridizing bands; a prominent mRNA of approximately 4.5 kb and two larger transcripts that hybridize less intensely (left panel; final wash: 0.1x SSC, 0.1% SDS, 50 C, exposure for 3 d at -80 C). Poly-A+ RNA from liver, kidney, and possibly heart revealed a single weakly hybridizing transcript that is approximately 4.5 kb in size, whereas poly-A+ RNA from liver showed an additional hybridizing band of about 1.5 kb (arrows), and poly-A+ RNA from brain showed evidence for transcripts of about 1 kb and possibly 0.7 kb (arrows) (right panel; 3-wk exposure).

 
To assess TIP39 expression further, in situ hybridizations were performed using those two tissues that express the PTH2 receptor most abundantly (e.g. brain and testis) and may thus represent targets of this peptide. Specific hybridization was detected in consecutive sections of adult mouse brain (Fig. 8Go, A–F), particularly within focal areas corresponding to the nucleus ruber and the nucleus centralis pontis, both of which have been implicated in the regulation of motor activity, and in the nucleus subparafascicularis thalami, which has been implicated in nociception. This distribution is different from the in situ data reported for the mRNA encoding the PTH2 receptor (13, 40), suggesting that TIP39 synthesis and secretion occurs distant from its site(s) of action. However, immunohistochemical studies may be necessary to determine whether TIP39—apart from possible paracrine/autocrine roles—is neuronally transported from the cerebral nuclei where it is synthesized to those areas of the brain, where the PTH2 receptor is expressed. Overall, in situ hybridizations and Northern blot analysis suggested that TIP39 is expressed in mice in only few tissues.



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  		Figure 8. TIP39 transcripts detected by RNA in situ hybridization using sagital sections of an adult mouse brain. A, Sagital section (H&E staining), corresponding to section 163 (43 ). The arrow depicts nucleus subparafascicularis thalami. B, Close-up (~x2) of the same section in dark field. C, Sagital section (H&E staining), roughly corresponding to section 143 (43 ); left arrow, nucleus ruber; right arrow, nucleus centralis pontis. D, Close-up (~x2) of the same section in dark field. E, Coronal section (H&E staining) [Bregma -2.92 mm (44 )], arrow depicting TIP39 expression in an area corresponding to nucleus subparafascicularis thalami. F, Close-up (~x2) of the same section in dark field. The bar represents 1 mm for sagital (A, C) and 0.5 mm for coronal (E) sections.

 
Most prominent TIP39 mRNA expression was detected in the epithelium of seminiferous tubules (Fig. 9Go). Analysis of the expression pattern suggested marked stage-specific differences. However, further studies are needed to assess the differentiation stage of those tubule segments expressing TIP39 mRNA. In contrast to these findings, PTH2 receptor expression in testis was reported to occur in the interstitium between spermatic tubules, i.e. in Leydig cells, as well as in sperm and within the epididymis (13). Taken together, these findings suggest that TIP39 and the PTH2 receptor may have a role in cAMP generation in seminiferous tubules and could thus have, similar to PACAP (42), a role in spermatogenesis. No hybridization of TIP39 mRNA was detected in pancreas, where the PTH2 receptor is also expressed (data not shown).



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  		Figure 9. TIP39 transcripts detected by RNA in situ hybridization in seminiferous tubuli. Representative section through an adult mouse testis (left panel, H&E staining; right panel, dark field; original magnification, x200) showing strong TIP39 expression in segments, which correspond to different stages of the spermatogenic cycle.

 
In summary, we provided information about the gene structure of the human and murine genes encoding TIP39. This peptide exhibited a distant phylogenetic relationship to PTH and PTHrP, and mouse and human TIP39 showed similar potency and efficacy in stimulating the human PTH2 receptor. While the biological role(s) of TIP39 is currently unknown, our findings are consistent with the notion that it is a neuroendocrine peptide, which may also be involved in the regulation of testicular function and/or differentiation.


   Footnotes
 
This work was supported by the National Institutes of Health (Grant DK-11794), the Deutsche Forschungsgemeinschaft (JO 315/1–2; to M.R.J.), and the Swedish Foundation for International Cooperation in Research and Higher Education (to K.B.J.).

1 Present address: Novartis Pharma AG, Bone Metabolism, Research Building K-125.10.59, CH-4002 Basel, Switzerland. Back

Abbreviations: dNTP, Deoxynucleoside triphosphate; GIP, gastrointestinal-inhibitory peptide; H&E, hematoxylin and eosin; NCBI, National Center for Biotechnology Information; ORF, open reading frame; PTH2 receptor, type 2 PTH receptor; RACE, rapid amplification of cDNA ends; TIP39, tuberoinfundibular peptide of 39 residues.

Received July 26, 2001.

Accepted for publication November 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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