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J Biol Chem, Vol. 274, Issue 33, 23035-23042, August 13, 1999

A G Protein-coupled Receptor from Zebrafish Is Activated by Human Parathyroid Hormone and Not by Human or Teleost Parathyroid Hormone-related Peptide
IMPLICATIONS FOR THE EVOLUTIONARY CONSERVATION OF CALCIUM-REGULATING PEPTIDE HORMONES^*

David A. Rubin^§, Per Hellman^¶, Leonard I. Zon, Craig J. Lobb^**, Clemens Bergwitz, and Harald Jüppner^§§¶¶

From the  Endocrine Unit, ^§§ Pediatric Services, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114,  Howard Hughes Medical Institute and Children's Hospital of Boston, Harvard Medical School, Boston, Massachusetts 02115, and ^** Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5' splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr³⁴]hPTH(1-34)-amide (hPTH) (EC₅₀, 1.64 ± 0.95 nM) and [Ile⁵,Trp²³,Tyr³⁶]hPTHrP-(1-36)-amide ([Ile⁵, Trp²³]hPTHrP) (EC₅₀, 46.8 ± 12.1 nM) but not when stimulated with [Tyr³⁶]hPTHrP-(1-36)-amide (hPTHrP), [Trp²³,Tyr³⁶]hPTHrP-(1-36)-amide ([Trp²³]hPTHrP), or [Ala²⁹,Glu³⁰,Ala³⁴,Glu³⁵,Tyr³⁶]fugufish PTHrP-(1-36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

In contrast to terrestrial vertebrates, fishes usually have an abundant supply of calcium but an insufficient supply of phosphate (1). In most fishes, the environmental challenges with regard to mineral ion homeostasis are therefore distinctly different than those in mammals, in which the regulation of calcium and phosphate is predominantly controlled by vitamin D, parathyroid hormone (PTH),¹ and a still poorly characterized phosphaturic factor (2-4). In mammals, PTH is expressed almost exclusively in the parathyroid glands, and its calcium-homeostatic actions are mediated through a G protein-coupled receptor, the type 1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R) (3, 5). In addition to mediating these endocrine actions of PTH, the widely expressed PTH1R also mediates, at least in mammals, the autocrine/paracrine actions of PTHrP (3, 6), which was first isolated from tumors of patients with the syndrome of humoral hypercalcemia of malignancy (7). PTHrP, similar to the PTH1R, is widely expressed (6) and is essential for normal endochondral bone formation (8), and most likely for a variety of other functions including tooth eruption and breast development (9-13).

PTH- and PTHrP-like immunoreactivity has been described in several fishes (2, 14-16). Partial DNA sequences encoding a putative fish PTH molecule were isolated from rainbow trout genomic DNA (17), and a genomic DNA sequence encoding a teleost PTHrP homolog was recently isolated from pufferfish (FUGU Landmark Mapping Project Database clones 115E01AC6 and 155E01eB5). However, the biological functions of PTH-like ligands in fishes remain largely elusive.

A second receptor for PTH, the PTH2-receptor (PTH2R), has highest homology to the PTH1R, but its biological role(s) remain to be determined (18, 19). Unlike the PTH1R, the PTH2R is activated by PTH and a partially characterized PTH-like peptide from the hypothalamus but not at all or very poorly by PTHrP (18-20). Furthermore, its expression is limited to a few tissues (18, 19), and due to this ligand specificity and its restricted tissue distribution, the PTH2R is unlikely to assume functions that are normally mediated by the PTH1R. Consequently, mice that lack the PTH1R die in utero or at birth (21) and show developmental defects that are similar, but more severe, than those observed for PTHrP-ablated animals (8).

To begin exploring the evolution of the receptors for PTH and PTHrP, we recently isolated two non-allelic cDNAs encoding PTH1Rs from the frog Xenopus laevis (22). To extend these studies and to begin evaluating the biological roles of these receptors in fishes, we have isolated partial genomic and cDNA clones encoding a G protein-coupled receptor from catfish (Ictalurus punctatus) and full-length cDNA clones encoding the zebrafish (Danio rerio) homolog. This receptor showed highest amino acid sequence identity with mammalian PTH2Rs, was activated by human PTH, but not by human or fugufish PTHrP, and is therefore likely to represent the zebrafish PTH2R (zPTH2R) homolog. Further characterization of the zPTH2R in vitro and in vivo will help define its biological role(s) in fishes and could provide insights into function(s) mediated by its mammalian homolog. The more detailed exploration of the evolution of PTH1Rs and PTH2Rs may furthermore lead to the identification of a common ancestral precursor and possibly other PTH/PTHrP-like receptors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Molecular Cloning of a cDNA Encoding Portions of the Catfish PTH2R-- Juvenile I. punctatus were obtained at a local aquarium, anesthetized with 2-phenoxyethanol (Sigma), and sacrificed. Liver and kidney were placed on dry ice and stored at 70 °C until total RNA was isolated using the guanidinium thiocyanate/-mercaptoethanol method (Stratagene, La Jolla, CA). Reverse transcription with Superscript II RNase H reverse transcriptase (Life Technologies, Inc.) was performed at 42 °C using primer A (5'-TGCTGTCAGAATAGGGACCTGGTA-3'; all nucleotides were synthesized by the MGH Polymer Core Facility) (Fig. 1) and total RNA from either catfish liver (10 µg) or kidney (7 µg). Forward and reverse primers for RT-PCR were based on previously isolated catfish genomic DNA encoding portions of a G protein-coupled receptor that showed highest amino acid sequence homology with the mammalian PTH2R (18, 23).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the cDNA encoding putative teleost PTH2 receptors. Vertical boxes depict the approximate location of the receptor portions that encode transmembrane (TM) domains 1-7; locations of the recognition sites for two endonucleases are shown;  indicates sites for potential N-linked glycosylation;  and  indicate the approximate location of primers that were used for RT-PCR, 3'- and 5'-RACE, and end-to-end PCR; the location of the probe for Southern blot hybridizations is indicated by .

A 50-µl PCR using primers A and C (5'-AACTACTACTGGATCCTGGTG-3') was performed using Life Technologies, Inc., Taq (5 units) on an MJ research thermal cycler (Watertown, MA); the following PCR profile was used for 35 cycles: initial denaturation at 95 °C for 3 min, denatured at 94 °C for 1 min, annealed at 58 °C for 1 min, and polymerized at 72 °C for 2 min. A nested PCR (24) with 3 µl from each of the initial liver- or kidney-derived RT-PCR products was performed with primers C and B (5'-CTTAATATTGCCTGCACTCAGCT-3') using the same PCR profile as before but for 40 cycles. PCR products were identified on an 1.5% agarose gel; DNA bands of approximately 300 bp were excised, purified by glassmilk (24), and ligated into pGEM-T (Promega, Madison, WI) for transformation of competent DH5 cells (Life Technologies, Inc.). Plasmid DNA was purified using standard protocols (24, 25) and sequenced by cycle sequencing (U. S. Biochemical Corp.) on an 8 M urea, 6% polyacrylamide field gradient gel (24, 25). The DNA sequences were analyzed by the GCG package program (26).

Molecular Cloning of cDNAs Encoding Full-length Zebrafish PTH2Rs-- Subsequent to the isolation of the partial catfish PTH2R (catfish PTH2R) cDNA clone, 3 µl of a zebrafish (D. rerio) random-primed kidney cDNA library in  ZAP (3 × 10⁶ pfu/ml) (27) were screened by PCR using primers A and C. PCR profile and conditions were the same as before, except that polymerization at 72 °C was extended to 3 min for 40 cycles, and a final 10-min extension at 72 °C was added. The zebrafish PCR product was excised, cloned, and sequenced as described above.

By using the  ZAP-specific anchor primers, forward SK (5'-CCGCTCTAGAACTAGTGGATC-3') or reverse RevT7 (5'-TTGTAATACGACTCACTATACGGC-3'), and several zPTH2R-specific primers (Fig. 1), two sets of nested PCRs were performed to isolate the 5' and 3' regions of the zPTH2R. Primers and conditions for 5' PCR are as follows: primers SK and A, 2 µl of the kidney cDNA library in a 100-µl PCR, using the same PCR profile as above; for 5'-nested PCR, primers SK and B, 1 µl from the SK-A product in a 100-µl PCR, and the same PCR profile above except that annealing was performed at 62 °C. Primers and conditions for 3'-PCR are as follows: primers C and RevT7, 2 µl of the kidney cDNA library in a 100-µl PCR, using the same PCR profile as above; for 3'-nested PCR, primers RevT7 and D (5'-ATGGCCTTCTTCTCGGACTCC-3'), 1 µl from the C-RevT7 product in a 100-µl PCR, and the same PCR profile as above except that annealing was performed at 62 °C. Both sets of nested PCRs were analyzed on a 1.5% agarose gel, and the DNA bands were excised and purified through spin columns (Bio 101, La Jolla, CA), ethanol-precipitated, and ligated into pGEM-T for transformation of DH5 cells, and sequenced as described above.

The thermostable proofreading DNA polymerase ULTma (Perkin-Elmer) was used with the zPTH2R-specific primer 5'UT (5'-CTGAGAATCGAAGGCCAAGAGG-3') and E (5'-TGCTACACCACTAGTCAGTCTATT-3') to amplify the entire coding region of the zPTH2R in a single end-to-end PCR (Fig. 1). The PCR product of approximately 1800 bp in length was excised and purified as described above and ligated into pcrBlunt (Invitrogen, Carlsbad, CA) to yield zPTH2R/pcrBlunt. Midiprep DNA (Qiagen, Valencia, CA) of the zPTH2R/pcrBlunt clone was prepared, and the entire clone was resequenced to confirm that it was identical to the previously isolated partial cDNA clones. To allow expression in COS-7 cells, the zPTH2R insert was excised by EcoRI (New England Biolabs, Beverly, MA) digestion and ligated into the corresponding site in pcDNAI/Amp (Invitrogen) to yield zPTH2R/pcDNAI/Amp.

Molecular Cloning of Putative Splice Variants of the zPTH2R-- By using a 5'-RACE kit (Life Technologies, Inc.) and total RNA from adult zebrafish, the 5' end of the cDNA encoding the zPTH2R was amplified with three successive reverse primers; these primers encode portions of the first membrane-spanning domain (TM1, 5'-AGAAACTGCATAGAAGACTGTGTACAT-3'), the distal (G, 5'-CCAGCACCGAGGAACCATTAG-3'), and a more proximal portion of the amino-terminal extracellular domain (E1, 5'-CCTTTTGGAGGCACTGCAGCTTTGC-3') (Fig. 1). The resulting cDNA clones were completely resequenced to determine their orientation and to confirm that they were identical to the previously isolated zPTH2R sequences. For expression studies, the BamHI-Eco57I fragment of one putative splice variant was ligated into zPTH2R/pcDNAI/Amp by replacing the corresponding BamHI-Eco57I fragment of zPTH2R, to yield zPTHrP(43) (Fig. 1).

Expression and Functional Evaluation of cDNAs Encoding Teleost and Mammalian Receptors for PTH and PTHrP in COS-7 Cells-- COS-7 African green monkey kidney cells were cultured as previously reported (28) in Dulbecco's modified Eagle's medium (DMEM, Mediatech, Washington, D. C.) supplemented with 10% fetal bovine serum (Sigma), 50 units/ml penicillin G, and 50 µg/ml streptomycin sulfate (Life Technologies, Inc.) at 37 °C in a humidified 95% air, 5% CO₂ atmosphere. COS-7 cells were transfected with plasmid DNA (300 ng/well in a 24-well plate) using the DEAE-dextran method as described (28). The transfected cells were cultured for 72 h at 37 °C, followed by an additional 24 h at 33 °C (29) until they were functionally evaluated after 96 h. 24-well plates containing transfected COS-7 cells (approximately 200,000 cells/well) were incubated for 1 h at room temperature in DMEM supplemented with 2 mM 3-isobutyl-1-methylxanthine, 0.1% bovine serum albumin, 20 mM HEPES (pH 7.4) in the absence or presence of increasing concentrations of either hPTH, hPTHrP, [Trp²³]hPTHrP, [Ile⁵,Trp²³]hPTHrP, or [Ala²⁹,Glu³⁰,Ala³⁴,Glu³⁵, Tyr³⁶]fugufish PTHrP-(1-36)-amide (fuguPTHrP) (FUGU Landmark Mapping Project Database clone 115E01AC6).² Peptides were synthesized by the Massachusetts General Hospital Polymer Core Facility as described (29); with the exception of fuguPTHrP, these peptides had been previously characterized with hPTH1R and hPTH2R (29). Cyclic AMP (cAMP) was determined by radioimmunoassay as described (28). All data points represent mean ± S.E. of two or more independent experiments performed in duplicate.

Southern Blot Analysis of Zebrafish Genomic DNA-- Approximately 10 µg of zebrafish genomic DNA was digested to completion with either EcoRI, HindIII, or BamHI (New England Biolabs, Beverly, MA). The samples were electrophoresed through a 0.8% agarose gel, transferred onto nitrocellulose membrane (Micron Separations, Inc., Westborough, MA), and baked in vacuo for 2 h at 80 °C. The DNA blots were hybridized (42 °C, 18 h) with a PCR-generated ³²P-labeled probe (30) encoding the carboxyl-terminal tail of the zPTH2R (411 bp; see Fig. 1) in a solution containing 50% formamide, 6× SSC, 5× Denhardt's solution, 0.1% SDS, 100 µg/ml sonicated calf thymus DNA, and 10% dextran sulfate. Washes were performed for 30 min each at room temperature with 1× SSC, 0.1% SDS, 50 °C in 1× SSC, 0.1% SDS, and a final wash at 55 °C in 1× SSC, 0.1% SDS; followed by autoradiography at 70 °C for 3 days with a DuPont Cronex intensifying screen and Kodak XAR film.

Phylogenetic and Structural Analyses of PTH1Rs and PTH2Rs-- Comparisons and alignments of the amino acid sequences of all known PTH1Rs and PTH2Rs were carried out by GCG analysis. Sequences were subsequently entered and aligned within MacClade 3.0 (31), with subsequent readjustments in the amino- and carboxyl-terminal regions to maximize the homology of the native proteins (32, 33). Each amino acid was treated as an unweighted character when analyzed using the branch-and-bound search option of PAUP 3.1 (34). The goldfish VIP receptor (GenBank^TM accession number U56391) and human CRF receptor, type A (GenBank^TM accession number P34998), were used as outgroup sequences. A bootstrapping analysis using the branch-and-bound option on 500 replicates (35, 36) was performed, and only groups that were compatible with the 50% majority-rule consensus were retained (34, 37).

The receptor regions for amino acid and nucleotide sequence comparisons (Table I) comprised the amino-terminal, extracellular domain (all sequences 5' of residue 172), the core regions of the receptors (all residues between amino acids 173 and 447), and carboxyl-terminal intracellular region (all residues 3' of residue 448); these numbers refer to the zPTH2R (Fig. 4).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Molecular Cloning of a Partial cDNA Encoding a Putative Catfish PTH2R-- A genomic DNA clone encoding portions of the putative PTH2R from channel catfish (I. punctatus) (initially thought to encode a teleost PTH1R homolog) had been previously isolated by screening a catfish genomic phage library with a probe encoding the rat PTH1R (23). Two exons had been identified which are, most likely, the equivalents of exons M3 and EL2 of the mammalian PTH/PTHrP receptor gene (38-40) (Fig. 2). RT-PCR using kidney and liver total RNA and primers based on the catfish genomic DNA sequence resulted in the isolation of identical 193-bp cDNA fragments from both tissues. These findings indicated that PTH2R expression in catfish may be different from that in mammals where, based on Northern blot analysis, PTH2R is expressed most abundantly in brain, pancreas, testis, and placenta (18). However, in situ hybridizations had previously shown that the PTH2R is expressed in mammalian kidney (19), and other more sensitive techniques may reveal additional tissues expressing this receptor. In addition to exons M3 and EL2 of the catfish PTH2R gene, the 193-bp RT-PCR product contained the equivalent of the mammalian exon M4, and primers based on this novel exonic sequence were used to establish the intron/exon borders for the catfish homolog of exon M4 (Fig. 2). Overall, the amino acid sequence encoded by the three catfish exons showed 83% amino acid homology when compared with the hPTH2R but only 72% when compared with the hPTH1R. This suggested that the partial genomic and cDNA clones from catfish encode a homolog of the mammalian PTH2R and not of the mammalian PTH1R.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Nucleotide and amino acid sequence alignment of the genomic clones encoding portions of the catfish PTH2R and the human PTH1R (40). Intronic nucleotide sequences are in lowercase letters; nucleotide sequences encoding exons M3, M4, and EL2 are shown in uppercase letters. Backslash marks denote intron/exon borders; ··· indicates nucleotide identity; - - - indicates amino acid identity; consensus nucleotides for splice donor/acceptor sites are underlined.

Interestingly, the genomic clone encoding portions of this putative catfish PTH2R showed, at least for the available exons, the same intron/exon borders, and the same exon length as described previously for the mammalian PTH1Rs (23, 38-40). The genes encoding both receptor subtypes therefore appear to have a similar intron/exon organization, and it is plausible that the genomic structure remained largely unchanged during the evolution from fish to mammals.

Molecular Cloning of a cDNA Encoding a Putative PTH2R from Zebrafish-- Although catfish (Teleostei: Division Ostariophysi) are useful for aquaculture and physiological studies, other ostariophysan fishes, such as zebrafish, have become preferred experimental animals for comparative genetics and developmental studies. We therefore decided to isolate the full-length zebrafish PTH2R, and screened a zebrafish kidney cDNA library by PCR using the catfish-specific primers A and C (27). After a 228-bp PCR product was shown to be closely related to the cDNA encoding the putative catfish PTH2R and the human and rat PTH2R (18, 19), a nested PCR approach was used to isolate overlapping cDNA clones that encode the entire zebrafish PTH2R homolog. The total length of the isolated putative zPTH2R transcript was 2429 bp, but a shorter PCR-generated receptor variant of 1743 bp, zPTH2R(43) (see below), was subsequently used for most expression studies (Fig. 1). The amino acid sequence identity between the zPTH2R and the available sequence of the catfish homolog was 93%.

As for the partial catfish PTH2R, the amino acid sequence encoded by the full-length zebrafish cDNA clone showed higher homology with the hPTH2R than with the hPTH1R (Fig. 3). Sequence identity between the zPTH2R and the human and rat receptor homologs was 63 and 60%, respectively, and 47 and 52% when compared with human and frog PTH1R, respectively, and less than 37% when compared with other members of this family of G protein-coupled receptors. The highest amino acid sequence conservation among the PTH2Rs was observed within the transmembrane region (inclusive of the extracellular and intracellular loops), whereas the carboxyl-terminal, intracellular tails, and the extracellular domains (including the signal peptide) were less well conserved (Table I). In addition to the overall amino acid sequence conservation, the putative zPTH2R contained, in contrast to the mammalian PTH1Rs (38), a similar polyadenylation signal as the mammalian receptor homologs (18, 19).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Alignment of teleost (catfish and zebrafish) and mammalian (rat and human) PTH2-receptors. Amino acid sequences of known PTH2Rs were aligned with the zebrafish homolog using GAP and/or pileup algorithms of the GCG package (26). Gaps were introduced to maximize sequence homology. Residues that are identical to the zPTH2R are represented by hyphens. Potential transmembrane domains are underlined, conserved cysteine residues in the extracellular regions are identified by #, and conserved consensus sites for potential N-linked glycosylation are denoted by *. The 17 residues that are not present in the putative splice variant, zPTH2R(43), are horizontally boxed; those residues that are likely to be specific for PTH2Rs are vertically boxed.

The region corresponding to exon E2 of the mammalian PTH1R genes was not present in the zPTH2R, which is similar to findings in the rat and human PTH2R homologs (18, 19) (Fig. 3). As previously shown, the frog PTH1Rs and all members of the PTH2R family lack the equivalent of exon E2 (22), and its deletion or modification in the rat PTH1R has been shown to have no major effect on receptor function (41). This implies that the amino acid sequence encoded by exon E2 of the mammalian PTH1Rs is a relatively recent evolutionary modification.

Identification of Putative Splice Variants of the zPTH2R-- Alternative splicing of mammalian PTH1Rs (42-46) and of other members of this receptor family (47-51) has been described, but most of these differently spliced receptors show impaired functional properties. Only some PTH1R variants in which the non-coding exons U1, U2, and U3 are alternatively spliced onto exon S are well expressed and fully functional (44, 46, 52). The signal peptide and significant portions of the amino-terminal, extracellular domain of the zPTH2R showed, in comparison to the corresponding regions of rat and human PTH2R, relatively poor amino acid sequence conservation (Fig. 3). Furthermore, the amino-terminal, extracellular region encoded by the most abundant zebrafish clone contained, immediately following the putative signal peptide, a 38-amino acid insertion that is not present in the human or rat PTH2R homolog. Therefore, we searched by 5'-RACE for splice variants of the zPTH2R with higher homology to the mammalian PTH2Rs in the amino-terminal region, and we obtained evidence for at least two alternatively spliced receptor variants.

One putative splice variant, zPTH2R(43), was identical to the initially isolated zPTH2R clone except that the insertion immediately following the signal peptide was 17 residues shorter (Fig. 3). It remains to be determined whether the amino acids that give rise to these different insertions (comprising either 38 or 21 residues) are encoded by one or two novel exons or whether this portion of the amino-terminal, extracellular domain of the zPTH2R is encoded by a single exon that is larger than that found in the mammalian genes. Since a similar insertion has been described for a corticotropin-releasing factor (CRF) (51), variations in length of this receptor region may also be present in several other members of this receptor family.

Another putative splice variant of the zPTH2R lacked the nucleotides immediately following the in-frame stop codon (6 codons upstream of the initiator AUG) and the nucleotides encoding the signal peptide (residues 89-270) (data not shown). The protein sequence encoded by this alternatively spliced receptor therefore started at residue 71 in the equivalent mammalian exon E1, which is similar to a splice variant (type III) of the rat PTH1R (44) and the goldfish VIP receptor (53). Similar to the mammalian PTH1Rs (42, 44, 45), the teleost PTH2R thus undergoes alternative splicing, and it appears plausible that similar pre-mRNA processing occurs with rat and human PTH2Rs.

Functional Expression of cDNAs Encoding the Zebrafish PTH2R in Mammalian COS-7 Cells-- When transiently expressed in COS-7 cells, plasmid DNA encoding zPTH2R(43) showed in response to PTH (10⁶ M) an approximately 3-fold increase in cAMP accumulation which is almost twice as high as the response observed with the predominant zPTH2R clone. Thus, although the maximal agonist-induced cAMP accumulation by COS-7 cells expressing zPTH2R(43) was poor (15.1 ± 2.3 pmol/well) in comparison to cells expressing the human PTH2R and PTH1R (281.5 ± 32.3 and 383.2 ± 22.9 pmol/well, respectively), the results are comparable to those obtained with cells stably expressing the goldfish VIP receptor (53). This relatively poor efficacy could be due to either poor expression of the teleost receptor protein in a mammalian cell line, poor coupling to the mammalian stimulatory G protein, or an ineffective interaction with mammalian receptor activity-modifying proteins (54), if these prove to be necessary for the function of PTH1Rs and PTH2Rs. COS-7 cells expressing either zPTH2R or zPTH2R(43) show no receptor-dependent cAMP accumulation in response to human or fugufish PTHrP (see below), human secretin (10⁶ M), salmon calcitonin (10⁶ M), and rat growth hormone-releasing factor (10⁶ M) (data not shown).

Because zPTH2R(43) showed better expression in mammalian COS-7 cells than the zPTH2R, the former teleost receptor variant was further characterized with mammalian PTH and PTHrP and with teleost PTHrP (Fig. 4A); hPTH1R and hPTH2R were used for comparison. Cells expressing the zPTH2R(43) had lower basal cAMP accumulation than the two mammalian PTH receptors (zPTH2R(43), 4.1 ± 0.8 pmol/well; hPTH2R, 23.9 ± 1.2 pmol/well; and hPTH1R, 10.0 ± 3.0 pmol/well). Activation of zPTH2R(43) was observed with hPTH (EC₅₀, 1.64 ± 0.95 nM) and with [Ile⁵,Trp²³]hPTHrP (EC₅₀, 46.8 ± 12.1 nM); however, in contrast to previous observations with cells expressing the hPTH2R (29), the latter PTHrP analog was only a partial agonist at the zebrafish receptor (data not shown). As with COS-7 cells expressing the hPTH2R, hPTHrP and [Trp²³]hPTHrP were inactive when tested with the zPTH2R(43), and cells expressing either the zebrafish or human PTH2R homolog showed no stimulation of cAMP accumulation when challenged with fuguPTHrP (Fig. 4, B and C). Thus, although the G protein-coupled receptor from zebrafish showed reduced efficiency and much lower efficacy than the hPTH2R, zPTH2R(43) had functional properties that are characteristic of a type 2 PTH receptor.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   A , alignment of the amino acid sequences of human PTH (hPTH) and PTHrP (hPTHrP) and of fugufish PTHrP (fuguPTHrP). B, functional analysis of COS-7 cells expressing the zPTH2R (43). C, the human PTH2R. D, the human PTH1R. Data for each receptor and the different peptides (, hPTH; , hPTHrP; , fuguPTHrP) are shown as mean ± S.E. of at least three independent transfections performed in duplicate and were normalized to the maximal ligand-dependent cAMP accumulation of each receptor.

Interestingly, fugufish PTHrP had a similar efficacy and only slightly reduced efficiency as hPTH and hPTHrP when tested with the hPTH1R (EC₅₀ values, 2.24 ± 0.10 nM for fuguPTHrP versus 0.36 ± 0.07 and 0.57 ± 0.01 nM for hPTH and hPTHrP, respectively) (Fig. 4D), indicating that PTHrP has maintained similar receptor specificity throughout evolution. It thus appears possible that PTH or a PTH-like peptide from teleosts may have functional characteristics that are similar to those of mammalian PTH, and the isolation of a PTH-selective receptor from zebrafish supports the notion that such a ligand does exist in fish and possibly other vertebrates that lack parathyroid glands.

Southern Blot Analysis Using a cDNA Probe Encoding the Carboxyl-terminal Portion of the zPTH2R-- Ploidy levels vary in teleosts, including the Siluriformes (catfish) and Cypriniformes (zebrafish) (55, 56). To support the notion that only one PTH2R gene is present in the zebrafish genome, Southern blot analysis was performed using zebrafish genomic DNA that had been digested with three infrequently cutting restriction endonucleases (Fig. 5). For each digest, only one genomic DNA fragment hybridized, under stringent conditions, to the cDNA probe encoding the carboxyl-terminal intracellular tail of the zPTH2R. Based on the intron/exon organization of the mammalian PTH1R, which appears to be similar to that of the teleost PTH2R (see Fig. 2), this probe is likely to be encoded by a single exon. The Southern blot data therefore indicate that the genome of this teleost species contains only a single PTH2R gene.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Southern blot analysis of zebrafish genomic DNA. A genomic DNA blot, prepared as described under "Materials and Methods," was hybridized with a probe encoding portions of the carboxyl-terminal, intracellular tail of the zPTH2R (see also Fig. 1). kb, kilobase.

Structural and Phylogenetic Comparison of PTH1Rs and PTH2Rs-- Despite considerable amino acid sequence variations when compared with the mammalian and non-mammalian members of the class II family of G protein-coupled receptors (3, 5), the zPTH2R comprised the same number of conserved extracellular cysteines, several other conserved "signature residues," and at least three conserved consensus sequences for potential N-linked glycosylation (Fig. 3).

Although the catfish PTH2R was not full length, there was sufficient amino acid sequence information to justify its inclusion in a cladistic analysis (32). Phylogenetic comparisons (34) with the aligned PTH2Rs and PTH1Rs indicated with statistical significance that the two receptor subtypes are, in the most parsimonious cladogram, sister groups (Fig. 6) and that the terminal branches of each node are congruent with the previously established, morphology-based phylogenies (57). Further analysis of the cladogram revealed 16 informative amino acids that change unambiguously for each branch, when compared with the PTH1Rs, and these residues are therefore likely to be characteristic of PTH2Rs (31, 32, 34). Through the analysis of types 1 and 2 receptors from additional species it may therefore be possible to identify invariant residues that are specific for each receptor subtype. Furthermore, it may be possible to predict whether a particular amino acid change can be of functional importance, as shown for mutations identified in rare genetic diseases in humans (58-61), and whether certain residues are likely to confer ligand binding and/or signaling specificity (28, 62).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Maximum parsimony analysis of the alignment of all known PTH1Rs (human, pig, rat, mouse, opossum, and Xenopus subtypes A and B) and PTH2Rs (zebrafish, catfish, rat, and human) using the Branch-and-Bound search option of PAUP 3.1 (34); the goldfish VIP receptor and human CRF receptor were used as outgroup sequences. Amino acid sequences were aligned by the pileup algorithm of the GCG package (26) with some manual adjustments as described (33); gaps were introduced to maximize amino acid sequence homology (PAUP 3.1). Each amino acid was treated as a character, and a total of 674 characters was evaluated. The most parsimonious tree had a length of 1287 steps and a consistency index of 0.913, if uninformative characters were excluded. The bootstrap confidence intervals are shown next to the branch points and indicate the percentage of trials that support a given branch in 500 branch-and-bound iterations.

In summary, two isolated zebrafish cDNAs encode full-length G protein-coupled receptors, and when expressed in COS-7 cells the zPTH2R(43) receptor variant is efficiently activated by human PTH but not by human or teleost PTHrP. Teleosts thus express a homolog of the mammalian PTH2R, which suggests that PTH or a PTH-like ligand appeared during evolution before parathyroid glands developed. The cDNAs from catfish and zebrafish represent important tools for exploring the biological importance of the teleost PTH2R and may prove helpful in the isolation of PTH-like ligands from this and possibly other non-mammalian vertebrate species.

	ACKNOWLEDGEMENTS

We thank Drs. Henry T. Keutmann, Tom Gardella, Ernestina Schipani, Henry M. Kronenberg, and John T. Potts, Jr., for their suggestions and comments.

	FOOTNOTES

^* This research was supported in part by National Institutes of Health Research Service Award F32 DK09500 (to D. A. R.) and National Institutes of Health Grants DK-11794.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) catfish PTH2R, AF132081; zebrafish PTH2R, AF32082; mouse PTH2R, AF132083; rat PTH1R, M77184; mouse PTH1R, X78936; human PTH1R, X68596; pig PTH1R, U18315; opossum PTH1R, M74445; Xenopus subtype A, 1204422; Xenopus subtype B, 1209822; rat PTH2R U55836; human PTH2R, U25128; goldfish VIP receptor, U56391; human CRF receptor, P34998.

^§ Recipient of National Research Service Award DK 09500.

^¶ Present address: Dept. of Surgery, University Hospital, Uppsala, Sweden.

Present address: Abteilung für Klinische Endokrinologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

^¶¶ To whom correspondence should be addressed. Tel.: 617-726-3966; Fax: 617-726-7543; E-mail: jueppner@helix.mgh.harvard.edu.

	ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; PTHrP, PTH-related peptide; PTH2R, PTH2-receptor; PTH1R, PTH/PTHrP receptor; DMEM, Dulbecco's modified Eagle's medium; hPTH, [Tyr³⁴]hPTH-(1-34)-amide; [Ile⁵,Trp²³]hPTHrP, [Ile⁵,Trp²³,Tyr³⁶] hPTHrP-(1-36)-amide; hPTHrP, [Tyr³⁶]hPTHrP-(1-36)-amide; [Trp²³] hPTHrP, [Trp²³,Tyr³⁶]hPTHrP-(1-36)-amide; fuguPTHrP, [Ala²⁹,Glu³⁰,Ala³⁴,Glu³⁵,Tyr³⁶]fugufish PTHrP-(1-36)-amide; exon M3, exon encoding transmembrane 3; exon M4, exon encoding transmembrane 4; exon EL2, exon encoding the second extracellular loop; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair; RACE, rapid amplification of cDNA ends; TM, transmembrane; CRF, corticotropin-releasing factor.

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