Figures
Abstract
Mammals adapted to a great variety of habitats with different accessibility to water. In addition to changes in kidney morphology, e.g. the length of the loops of Henle, several hormone systems are involved in adaptation to limited water supply, among them the renal-neurohypophysial vasopressin/vasopressin receptor system. Comparison of over 80 mammalian V2 vasopressin receptor (V2R) orthologs revealed high structural and functional conservation of this key component involved in renal water reabsorption. Although many mammalian species have unlimited access to water there is no evidence for complete loss of V2R function indicating an essential role of V2R activity for survival even of those species. In contrast, several marsupial V2R orthologs show a significant increase in basal receptor activity. An increased vasopressin-independent V2R activity can be interpreted as a shift in the set point of the renal-neurohypophysial hormone circuit to realize sufficient water reabsorption already at low hormone levels. As found in other desert mammals arid-adapted marsupials show high urine osmolalities. The gain of basal V2R function in several marsupials may contribute to the increased urine concentration abilities and, therefore, provide an advantage to maintain water and electrolyte homeostasis under limited water supply conditions.
Citation: Böselt I, Römpler H, Hermsdorf T, Thor D, Busch W, Schulz A, et al. (2009) Involvement of the V2 Vasopressin Receptor in Adaptation to Limited Water Supply. PLoS ONE 4(5): e5573. https://doi.org/10.1371/journal.pone.0005573
Editor: Leslie B. Vosshall, The Rockefeller University, United States of America
Received: February 23, 2009; Accepted: April 6, 2009; Published: May 18, 2009
Copyright: © 2009 Böselt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (SFB 610), Bundesministerium fuer Bildung und Forschung and Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Leipzig. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Over a period of 170 million years of mammalian evolution species adapted to a great variety of habitats with different accessibility to water. Several mammals managed adaptation to conditions of water restriction among them desert rodents and elephants. Interestingly, elephants still present anatomical and physiological features of their evolutionary origin as aquatic animals [1], [2]. Other closely related mammals such as Baikal and California seals managed adaptation to fresh and sea water conditions, respectively. There is strong evidence that, besides morphological modifications in the kidney [3], [4], several hormone systems [5], among them the renal-neurohypophysial hormone system of vasopressin, are involved in adaptation to differences in fresh water supply [6].
The peptides of the vasotocin/vasopressin/oxytocin family are evolutionary old hormones which are already found in agnate bony and cartilaginous fishes [7]–[9]. At least one homolog each of vasopressin and oxytocin (mesotocin, isotocin) is found in jawed vertebrates whereas jawless vertebrates possess vasotocin [10]. Along with the divergence of the vasopressin/oxytocin peptides their receptors developed co-evolutionary and in mammals at least two of the various vasopressin receptor subtypes and one oxytocin receptor became established within all the genomes investigated so far. All members of the vasopressin receptor family belong to the superfamily of rhodopsin-like G protein-coupled receptors (GPCR). In mammals V1 vasopressin receptors (V1aR, V1bR) are involved in blood pressure regulation and central feedback mechanism whereas the V2 vasopressin receptor (V2R) maintains the water balance and electrolyte homeostasis.
The V2R is mainly expressed in kidney and regulates aquaporin-2-mediated water reabsorption via activation of the Gs protein/adenylyl cyclases/cAMP pathway. Vasopressin receptor function specialized in the regulation of water and electrolyte homeostasis is obviously linked to the evolution of tetrapods [11] but there is evidence that vasopressin-regulated cAMP formation is already present in fishes [12].
The importance of the vasopressin/V2R system in the hypothalamic-renal regulation of the water and electrolyte homeostasis becomes evident when one of the components is inactivated. For example, inactivating mutations of the V2R gene cause an inherited form of nephrogenic diabetes insipidus (NDI) [13]. NDI is characterized by the inability to concentrate the urine and an increased urine volume. Inherited NDI is not only found in humans but also in mice, dogs and horses [14]–[16] indicating similar functional relevance of the V2R among mammals. However, there are several examples where patients managed NDI basically by appropriate water supply (family 4 in [17] and family 1 in [18]). This may suggest that partial or even full loss of V2R function is tolerated in species with free access to fresh water.
An opposite phenotype, the nephrogenic syndrome of inappropriate antidiuresis (NSIAD), is seen when V2R is altered by activating mutations. Here, the phenotype is characterized by serum hypoosmolarity and high urinary sodium levels [19]. This may suggest that increased activity of V2R provides some advantage in concentrating the urine in order to save water under dry environmental conditions. One can speculate that the limited alimentary water supply prevents serum hypoosmolarity in desert animals, a sign found in patients with NSIAD under water-ad-libitum conditions.
According to allosteric ternary complex models of receptor activation [20], V2R, like all GPCR, exists in an equilibrium between inactive and active conformations. The models predict that, even in the absence of agonist, a certain fraction of receptors will spontaneously adopt an active conformation, permitting agonist-independent G-protein activation. Agonist binding to the free receptor leads to stabilization of the activated form of the receptor that is able to couple to the G protein. Inverse agonists decrease the spontaneous activity of receptors by shifting the equilibrium towards the inactive stage. Like agonists or inverse agonists, activating or inactivating mutations are capable of shifting the equilibrium toward active and inactive conformations, respectively. The fact that structural variations of the V2R may have an impact on basal activity, efficacy and potency gives rise to a spectrum of adaptive functionalities.
Here, we set out to analyze whether structural and functional properties of mammalian V2R correlate to different environmental conditions. We focused on V2R rather than on the hormone vasopressin because it is also involved in blood pressure regulation and the latter physiological function may restrict the adaptive variability of the peptide hormone. Thus, 87 V2R ortholog sequences were retrieved from mammalian species, some existing in extreme habitats. This sequence information enabled us to identify motifs and residues that are important for maintaining the receptor function. Our functional studies provide evidence that increased basal V2R activity is probably responsible for increased basal urine concentration ability in some desert-living marsupials.
Results and Discussion
Public sequence databases were mined for mammalian genomic V2R sequences. We further increased the sequence information by fully or partially cloning of additional V2R orthologs (see Supporting Material Table S1) with special focus on mammalian species living in desert environments and habitats with water excess. The sequence information of 87 full-length or partially identified V2R orthologs enabled us to determine and to compare structural parameters of the V2R gene and protein which are relevant for maintaining receptor's function.
Preservation of the genomic structure of the V2R in mammals
Previous studies have shown that some structural variability of V2R is generated by receptor splice variants e.g. in rodents [21]–[23]. Further, mutations of splice sites within the V2R gene (AVPR2) were found to be the cause of inherited NDI [24]–[27]. Therefore, we analyzed the genomic structure of mammalian V2R genes in respect to the conservation of splice donor and acceptor sites.
Three exons encode for the human V2R [28] and this genomic structure appears to be evolutionary preserved among mammals (see Figure 1). The second intron is ancient and is found not only in V2R genes of birds and mammals but also in other members of the vasopressin/oxytocin receptor family at the same position [29], [30]. The first intron was probably introduced in the very early mammalian evolution because it is found in platypus, marsupial and other mammalian but not in avian V2R genes.
To determine the conservation of the genomic structure, the sequence of 87 mammalian V2R genes were retrieved by cloning from genomic DNA and by mining public databases. During 170 million years of mammalian evolution the genomic structure of three coding exons was preserved. There is experimental evidence that the V2R gene of several mammals including the human V2R gene contains an additional non-coding exon in 5′ position (not shown). Sequence alignments of the exon/intron boundaries indicate high conservation of the splice donor and acceptor sites. The positions of NDI-causing splice site mutations are marked with an asterisk.
Although the intron sizes vary between 117 bp and 757 bp for intron 1 and 75 bp and 534 bp for intron 2, the core sequences of the donor and acceptor sites in mammalian V2R genes are highly conserved (see Figure 1). All three mutations in the human V2R proposed to affect pre-mRNA splicing [31] are at splice site positions that are 100% conserved during mammalian evolution.
High structural conservation of V2R in mammals
The overall identity between full-length mammalian V2R orthologs (36 species) is 85.9±6.7% (see Table 1). During 170 million years of mammalian evolution ∼160 (∼43.1%) out of all amino acid residues in V2R remained unchanged between mammalian species (Figure 2). The overall structural conservation of V2R (84.6±8.0%) is not significantly different when compared with comparable ortholog data sets of other GPCR (20 identical species): rhodopsin (94.3±2.2%), the melanocortin type 4 receptor (MC4R) (93.1±4.8%), the ADP receptor P2Y12 (88.8±5.3% ) and GPR34 (85.4±7.5% ) [32]–[34].
The amino acid sequence of the human V2R is shown. Positions conserved in mammalian V2R orthologs are depicted in dark grey. Positions which vary only by two amino acids are shown in light gray. Positions given in white are not preserved in mammals. For the N-terminal part (position 1 to 64, numbering refers to the human V2R amino acid sequence) we analyzed 38 ortholog sequences, for the middle part (position 65 to 322) 87 sequences and for the C-terminal part (position 323–371) 42 sequences. Over 103 missense mutations were identified in NDI patients, most affected highly conserved amino acid residues (positions are encircled in black) (see also Table 3).
Detailed analysis revealed the following results:
First, we found several length variations within the extracellular and intracellular loops (see Table 1). The length variations of the third intracellular loop (ICL3) were most prominent (Figure 3). Although ICL3 has been implicated in G-protein activation, desensitization and interaction with other signalling components in other GPCR, ICL3 of the V2R shows low amino acid sequence conservation. In several species the middle part of ICL3 contains sequence deletions up to 11 amino acids (marsupials) and insertions of 16 amino acids (platypus) when compared to the human V2R. This implicates a rather low functional relevance of the middle portion of the ICL3 in V2R. Indeed, deletion of the respective 11 amino acids in the human V2R (referred to as human del243–253) had no effect on receptor function (Table 2). Additional evidence for a rather low functional relevance of the middle portion of ICL3 comes from split receptors. V2R fragments split in ICL3 reassemble functionally when coexpressed [35]. Further, a naturally occurring 12-bp deletion including amino acid positions 247 to 250 was reported to have no functional relevance in the human V2R [36].
Sequence analysis of V2R orthologs revealed a remarkable length and amino acid variation of ICL3. Positions which differ from the amino acid sequence of human V2R are boxed.
Second, the N terminus is most diverse in its length and amino acid sequence between mammalian orthologs (Table 1) clearly indicating less specific relevance in ligand binding and in receptor activity.
Third, the C terminus of the human V2R contains two Cys residue (Cys341, Cys342) which allows anchoring via palmitoylation and forming a fourth intracellular loop (ICL4). It was demonstrated by mutagenesis of the two Cys residues that palmitoylation of V2R is important for intracellular trafficking and/or sequestration/internalization. Most efficient reduction of receptor cell surface expression was found when Cys341 alone or both Cys residues were mutated [37]. Consistently, Cys at position 341 is 100% conserved in mammals whereas Cys342 is substituted by Arg, Trp and Phe in several mammalian species.
Fourth, a proposed glutamate/dileucine motif equivalent (E335LRSLL340 in human V2R) in the C terminus [38] is highly preserved during mammalian V2R evolution. The motif may represent a transport signal from the endoplasmic reticulum to the Golgi apparatus. Leu339 was found substituted by Trp and Leu340 by Phe in some V2R orthologs keeping with the hydrophobic nature of ICL4 [39].
Fifth, most rhodopsin-like GPCR possess an evolutionarily conserved Asp-Arg-Tyr (DRY) motif in the C-terminal region of TMD3. Mutations of the first two residues within this motif usually alter receptor function and, when they occur naturally, can even cause diseases. In V2R the DRY motif is a DRH which is highly conserved in mammals and only the manatee has DRQ. Variation of the Tyr residue in the DRY motif appears to be functionally tolerated since this position is variable in many GPCR and substitution of Tyr has no or marginal effects in most cases [40].
Sixth, phosphorylation plays a pivotal role in the regulation of GPCR function. Previous studies identified putative sites which are clustered in the C terminus and in ICL3 and are phosphorylated by specific GPCR kinases (GRK) and by protein kinase A (PKA), respectively [41]–[43]. Whereas abundant putative Ser/Thr phosphorylation sites are found in the C terminus of all V2R orthologs the putative PKA phosphorylation site within ICL3 at Ser255 [41] is substituted by Asn, Gly and Asp in many V2R orthologs implicating that this position is, if at all, a substrate for PKA in only a few species.
Evaluation of naturally occurring mutations in the human V2R
As stated above, several NDI patients carrying an inactivating V2R mutation managed the disease by sufficient fresh water supply without any medications. One can speculate that V2R function is less important when a species lives in a water-ad-libitum environment. Therefore, we analyzed whether NDI-causing (inactivating) variants of the human V2R naturally occur in other mammalian V2R orthologs. To date, about 113 missense variants of the human V2R at 90 amino acid positions have been described (see Supporting Material Table S3) [31], [44]. Out of these missense variants 91% are assumed to be NDI causing. We correlated this information with the evolutionary variability at the respective amino acid position (Figure 2, Table 3). About 91% of all NDI-causing mutations hit positions that are fully conserved or vary between two amino acids (Table 3). None of the NDI-causing variants was found in any of the mammalian V2R orthologs. Therefore, our analysis provided no evidence of inactivating mutations in the mammalian V2R orthologs investigated.
Adaptive gain-of-function in marsupial V2R orthologs
Conservation and variability of distinct positions can only be properly interpreted in the light of functional data of different orthologs. Therefore, a number of mammalian V2R orthologs were cloned, expressed in COS-7 cells, stimulated with arginine vasopressin (AVP) and functionally analyzed in cAMP assays. As shown in Table 2, AVP potencies (EC50) at the selected V2R orthologs varied between 115 pM (Californian sea lion) and 841 pM (dog) and were in a comparable range to findings with the human V2R (238 pM). There is no obvious correlation of the agonist potencies to the environmental conditions of the respective species. Our data may reflect the naturally tolerated range of vasopressin potency between roughly 0.1 and 1 nM. Consistently, there is no report where an exclusive increase in EC50 values of lower than one order of magnitude causes NDI.
Species differences were found for the efficacy of receptor signaling. Several aquatic mammals (Baikal seal, Californian sea lion, minke whale, Caribbean manatee), cattle and some gerbils (Wagner's gerbil, bushy-tailed jird) displayed reduced Emax values when compared with the human V2R (see Table 2). The reduced Emax values are most likely caused by reduction in cell surface expression levels of these V2R orthologs. For the Baikal seal V2R, one can discuss this as some reduction of constraint because of free access to fresh water and, therefore, as a reduced necessity to concentrate the urine. However, the reduced efficacy of V2R signal transduction in marine mammals (Californian sea lion, minke whale, Caribbean manatee) and gerbils is unexpected because these animals produce highly concentrated urine, which is especially important for mammals in a hyperosmotic and desert environment, respectively. Variations in kidney morphology observed in marine mammals does not appear to afford them any greater benefit than terrestrial mammals, suggesting that the adaptation of mammals to sea water was accomplished also via hormonal regulation of urine concentration and/or the rate of urine formation [45]. The anti-diuretic function of AVP in marine mammals is still inconclusive. AVP levels in dolphins and seals are rather low despite high urine osmolality [45]. In sum, it is unlikely that the reduced signaling efficacy of V2R in some aquatic mammals and gerbils is a result of evolutionary adaptation. It rather reflects the naturally occurring spectrum of V2R function or a reduced constraint at the V2R because of other adaptive mechanisms to concentrate the urine.
Striking differences were found in the basal activity of marsupial V2R orthologs. All marsupial V2R showed elevated basal activity ranging from 2.2 to 4.3-fold over the human and all other V2R orthologs tested (Figure 4A, Table 2). The orthologs of arid-adapted species, red kangaroo and agile wallaby, displayed the highest basal activity and a significant gain in receptor efficiency. ELISA studies exclude that the increased basal activity was due to higher cell surface expression levels. Basal cAMP levels displayed strong correlation to the transfected plasmid DNA indicating genuine constitutively active receptors (Figure 4B). The increase in basal activity of e.g. the red kangaroo V2R was comparable to basal cAMP levels found in patients with NSIAD [19]. Besides basal activity, marsupial V2R displayed normal V2R function and cellular expression levels (Table 2). When compared to all other mammalian V2R orthologs tested the kangaroo V2R contains 23 positions with amino acids that are unique to marsupial orthologs (see Supporting Material Figure S2). Thirteen of them are at positions which are 100% conserved among all other non-marsupial V2R orthologs. Unfortunately, there is no obvious individual position known to cause constitutive activity when substituted. It is more likely, that sequential or combinatorial substitutions at the sequence background of the marsupial V2R orthologs lead to increased basal receptor function. This hypothesis is supported by the fact that constitutive activity differed significantly between marsupial V2R orthologs (see Table 2).
For functional characterization of the human and marsupial V2R orthologs, the respective expression plasmids were transfected into COS-7 cells and tested for AVP-induced cAMP accumulation. A) 48 hours after transfection cells were stimulated with increasing concentrations of AVP. Intracellular cAMP was measured with AlphaScreen cAMP assay (see Materials and Methods). B) To further assess the increased basal activity of marsupial V2R orthologs increasing amounts of plasmid DNA from the red kangaroo ortholog were transfected and cAMP assays were performed. As expected for a constitutive active receptor, basal receptor activity correlates with the amount of transfected plasmid DNA. Data are given as mean±S.E.M. of three experiments each performed in duplicate.
Several marsupials, like the red kangaroo and the agile wallaby, are adapted to extremely dry climates. Most marsupials display a typical mammalian pattern of hormonal control of kidney function and water excretion, with plasma vasopressin levels correlating highly with the urine/plasma osmolality ratio [46]. However, their renal ability to reabsorb water and concentrate the urine is extraordinary high. For example, hare wallaby and red kangaroo have average urine osmolalities between 1,843 and 2,357 mosmol kg−1 [47] and red kangaroo can concentrate up to 4,000 mosmol kg−1 [48]. For comparison, average human urine osmolality is 800 mosmol kg−1 and human kidneys can concentrate the urine up to 1,400 mosmol kg−1. Although some studies suggest that mammals with relatively long loops of Henle for their body size tend to have greater than average urinary concentrating ability [49], [50], detailed analyses found no relationship between urine osmolality and absolute length of the loop of Henle [4], [51]. Moreover, a recent report found that one desert wallaby (Petrogale rothschildi) appears to be unique amongst mammals lacking antidiuretic response to vasopressin [47]. Therefore, other factors than morphological and hormonal must contribute to adaption of the renal water reabsorption to the different water accessibilities. An increased agonist-independent V2R function, as found for marsupials, can be interpreted as a shift in the set point of the renal-neurohypophysial hormone circuit to realize sufficient water reabsorption already at low hormone levels. Similarly, increased basal activity is physiologically found also for the thyroid stimulating hormone (TSH) receptor, the MC1R and the MC4R to assure basal thyroid hormone production, pigmentation and energy homeostasis, respectively [52]–[54]. One may argue that increased basal V2R activity in marsupials should have pathophysiological consequences as found in NSIAD patients. However, serum hypoosmolality in these patients results from normal alimentary water intake but inadequate renal water elimination. In contrast to humans one can not expect an ad libitum supply of water for desert marsupials. Therefore, serum hypoosmolality, as observed in NSIAD patients carrying an activating V2R mutation, is probably unlikely. This example nicely demonstrates the biological interpretation of a specific protein function (e.g. basal activity) as an advantage (adaptation) or a disadvantage (disease) depending on environmental conditions.
Conclusion
The V2R is a key component in regulating the renal water reabsorption but its contribution to adaptation of mammals to different water accessibility was not investigated yet. We found that the mammalian V2R is highly conserved in its amino acid structure and functional properties. Although some mammals have unlimited access to fresh water there is no evidence for complete loss of V2R function in those or any other mammals investigated. This indicates that some V2R activity is essentially required for species survival. In contrast, structural changes in the marsupial V2R resulted in a gain of V2R function and may provide an advantage to maintain water and electrolyte homeostasis for those marsupials living in arid habitats.
Materials and Methods
V2 vasopressin receptor ortholog identification and site-directed mutagenesis
To analyze the sequence of V2R orthologs, genomic DNA samples were prepared from tissue or peripheral mononuclear blood cells of various mammalian species (sources are given in Supporting Material Table S1). Tissue samples were digested in lysis buffer (50 mM Tris/HCl, pH 7.5, 100 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg/ml proteinase K) and incubated at 55°C for 18 hours. DNA was purified by phenol/chloroform extraction and ethanol precipitation. Degenerated primer pairs (Supporting Material Table S2) were applied to amplify V2R specific sequences. PCR reactions were performed with Taq polymerase under variable annealing and elongation conditions. Specific PCR products were directly sequenced and/or subcloned for sequencing into the pCR2.1-TOPO vector (Invitrogen, La Jolla, CA). Sequencing reactions were performed with a dye-terminator cycle sequencing kit (Applied Biosystems) on an ABI 3700 automated sequencer (Applied Biosystems). Based on considerable sequence similarities of the 5′- and 3′-untranslated regions (UTR) of V2R genes primers were designed (Supporting Material Table S2) which allowed for the identification of some sequences encoding the N and C termini of mammalian V2R orthologs.
Full-length V2R were inserted into the mammalian expression vector pcDps (Schöneberg et al 1996) and epitope-tagged with an N-terminal hemagglutinin (HA) epitope and a C-terminal FLAG-tag by a PCR-based overlapping fragment mutagenesis approach. For most species only genomic DNA was available for V2R ortholog cloning. Therefore, most mammalian V2R constructs were build by inserting a genomic DNA fragment containing the second intron (encompassing amino acid position 68 (2.33, numbering of the relative position within GPCR [55]) to amino acid position 321 (7.49) of the respective mammalian ortholog into the double-tagged human V2R-pcDps expression plasmid keeping the human N and C termini. Identity of all constructs and correctness of all PCR-derived sequences were confirmed by restriction analysis and sequencing. To assure that these constructs are functionally equivalent to expression constructs of the full-length cDNA, the different constructs were compared in cAMP assays. As shown in Supporting Material Figure S1 function of cDNA constructs (dog, bovine) were identical to constructs containing the TMD core and second intron of the respective species flanked by the human N and C termini.
Cell culture and functional assays
COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified 7% CO2 incubator. Lipofectamine™ (Invitrogen) was used for transient transfection of COS-7 cells. Thus, cells were split into 50-ml cell culture flasks (1×106 cells per flask) and transfected with 4 µg of plasmid DNA/flask.
ALPHAScreen™ cAMP assay.
cAMP content of cell extracts was determined by a non-radioactive cAMP accumulation assay based on the ALPHAScreen™ technology according to the manufacturer's protocol (Perkin Elmer LAS, Rodgau-Jügesheim, Germany) [56]. One day after transfection cells were split into 48-well plates (5×104 cells/well). Stimulation with various agonist concentrations ([Arg8]-vasopressin acetate salt from Sigma-Aldrich, Seelze, Germany) was performed 48 h after transfection for 1 h at 37°C. Reactions were stopped by aspiration of media and cells were lysed in 50 µl of lysis buffer containing 1 mM 3-isobutyl-1-methylxanthine. From each well 5 µl of lysate were transferred to a 384-well plate. Acceptor beads (in stimulation buffer without 3-isobutyl-1-methylxanthine) and donor beads were added according to manufacturers' protocol. Cyclic AMP accumulation data were analyzed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, California, USA).
ELISA studies.
To estimate cell surface expression of receptors carrying an N-terminal HA-tag, we used an indirect cellular ELISA [57]. To further assess the amounts of full-length double-tagged V2Rs, a sandwich ELISA was used essentially as described previously [17]. Briefly, microtiter plates were coated with a polyclonal anti-FLAG antibody and cell lysates were applied. Following intensive washing with PBS containing 0.05% Triton X-100 (PBS-T), plates were incubated with a peroxidase-labeled monoclonal anti-HA antibody (12CA5; 1 mg/mL PBS-T). After removal of excess unbound conjugate, H2O2 and o-phenylenediamine (2.5 mmol/L each in 0.1 mol/L phosphate/citrate buffer, pH 5.0) were added to serve as substrate and chromogen, respectively. After 15 min, the enzyme reaction (carried out at room temperature) was stopped by the addition of 1 mol/L H2SO4 containing 0.05 mol/L Na2SO3, and colour development was measured bichromatically at 492 and 620 nm using an ELISA reader (Sunrise™, Tecan Group Ltd.).
Data deposition
The sequences reported in this paper have been deposited in the GeneBank database (accession no. FJ411185-FJ411251; Supporting Material Table S1).
Supporting Information
Figure S1.
Equivalence of cDNA and TMD core constructs.
https://doi.org/10.1371/journal.pone.0005573.s001
(0.12 MB PDF)
Figure S2.
Structural differences between marsupial and non-marsupial V2R orthologs.
https://doi.org/10.1371/journal.pone.0005573.s002
(0.43 MB PDF)
Table S1.
Description, database accession and sources of genomic DNA samples, where applicable.
https://doi.org/10.1371/journal.pone.0005573.s003
(0.04 MB PDF)
Table S2.
Primers used for V2 ortholog amplification and cloning.
https://doi.org/10.1371/journal.pone.0005573.s004
(0.01 MB PDF)
Table S3.
Missense mutations found in human V2R.
https://doi.org/10.1371/journal.pone.0005573.s005
(0.01 MB PDF)
Acknowledgments
We would like to thank Christian Pitra, Christian Kern and the numerous contributors for the species samples (Supporting Material Table S1). We are grateful to Claudia Stäubert and the reviewers for many suggestions and critical reading of the manuscript.
Dedicated to Charles Darwin's bicentenary
Author Contributions
Conceived and designed the experiments: IB HR TS. Performed the experiments: IB HR TH WB. Analyzed the data: IB HR TS. Contributed reagents/materials/analysis tools: TH DT AS. Wrote the paper: IB TS.
References
- 1. Liu AG, Seiffert ER, Simons EL (2008) Stable isotope evidence for an amphibious phase in early proboscidean evolution. Proc Natl Acad Sci U S A 105: 5786–5791.
- 2. Gaeth AP, Short RV, Renfree MB (1999) The developing renal, reproductive, and respiratory systems of the African elephant suggest an aquatic ancestry. Proc Natl Acad Sci U S A 96: 5555–5558.
- 3. Mbassa GK (1988) Mammalian renal modifications in dry environments. Vet Res Commun 12: 1–18.
- 4. Beuchat CA (1996) Structure and concentrating ability of the mammalian kidney: correlations with habitat. Am J Physiol 271: R157–179.
- 5. McCormick SD, Bradshaw D (2006) Hormonal control of salt and water balance in vertebrates. Gen Comp Endocrinol 147: 3–8.
- 6. Bradshaw D (2007) Environmental endocrinology. Gen Comp Endocrinol 152: 125–141.
- 7. Acher R (1996) Molecular evolution of fish neurohypophysial hormones: neutral and selective evolutionary mechanisms. Gen Comp Endocrinol 102: 157–172.
- 8. Suzuki M, Kubokawa K, Nagasawa H, Urano A (1995) Sequence analysis of vasotocin cDNAs of the lamprey, Lampetra japonica, and the hagfish, Eptatretus burgeri: evolution of cyclostome vasotocin precursors. J Mol Endocrinol 14: 67–77.
- 9. Acher R, Chauvet J, Chauvet MT, Rouille Y (1999) Unique evolution of neurohypophysial hormones in cartilaginous fishes: possible implications for urea-based osmoregulation. J Exp Zool 284: 475–484.
- 10. Gwee PC, Amemiya CT, Brenner S, Venkatesh B (2008) Sequence and organization of coelacanth neurohypophysial hormone genes: evolutionary history of the vertebrate neurohypophysial hormone gene locus. BMC Evol Biol 8: 93.
- 11. Takei Y, Kawakoshi A, Tsukada T, Yuge S, Ogoshi M, et al. (2006) Contribution of comparative fish studies to general endocrinology: structure and function of some osmoregulatory hormones. J Exp Zoolog A Comp Exp Biol 305: 787–798.
- 12. Perrott MN, Sainsbury RJ, Balment RJ (1993) Peptide hormone-stimulated second messenger production in the teleostean nephron. Gen Comp Endocrinol 89: 387–395.
- 13. Fujiwara TM, Bichet DG (2005) Molecular biology of hereditary diabetes insipidus. J Am Soc Nephrol 16: 2836–2846.
- 14. Luzius H, Jans DA, Grunbaum EG, Moritz A, Rascher W, et al. (1992) A low affinity vasopressin V2-receptor in inherited nephrogenic diabetes insipidus. J Recept Res 12: 351–368.
- 15. Schott HC 2nd, Bayly WM, Reed SM, Brobst DF (1993) Nephrogenic diabetes insipidus in sibling colts. J Vet Intern Med 7: 68–72.
- 16. Yun J, Schoneberg T, Liu J, Schulz A, Ecelbarger CA, et al. (2000) Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene. J Clin Invest 106: 1361–1371.
- 17. Pasel K, Schulz A, Timmermann K, Linnemann K, Hoeltzenbein M, et al. (2000) Functional characterization of the molecular defects causing nephrogenic diabetes insipidus in eight families. J Clin Endocrinol Metab 85: 1703–1710.
- 18. Schulz A, Sangkuhl K, Lennert T, Wigger M, Price DA, et al. (2002) Aminoglycoside pretreatment partially restores the function of truncated V(2) vasopressin receptors found in patients with nephrogenic diabetes insipidus. J Clin Endocrinol Metab 87: 5247–5257.
- 19. Feldman BJ, Rosenthal SM, Vargas GA, Fenwick RG, Huang EA, et al. (2005) Nephrogenic syndrome of inappropriate antidiuresis. N Engl J Med 352: 1884–1890.
- 20. Lefkowitz RJ, Cotecchia S, Samama P, Costa T (1993) Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci 14: 303–307.
- 21. Sangkuhl K, Schulz A, Rompler H, Yun J, Wess J, et al. (2004) Aminoglycoside-mediated rescue of a disease-causing nonsense mutation in the V2 vasopressin receptor gene in vitro and in vivo. Hum Mol Genet 13: 893–903.
- 22. Sarmiento JM, Anazco CC, Campos DM, Prado GN, Navarro J, et al. (2004) Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant. J Biol Chem 279: 47017–47023.
- 23. Firsov D, Mandon B, Morel A, Merot J, Le Maout S, et al. (1994) Molecular analysis of vasopressin receptors in the rat nephron. Evidence for alternative splicing of the V2 receptor. Pflugers Arch 429: 79–89.
- 24. Kamperis K, Siggaard C, Herlin T, Nathan E, Hertz JM, et al. (2000) A novel splicing mutation in the V2 vasopressin receptor. Pediatr Nephrol 15: 43–49.
- 25. Wildin RS, Cogdell DE, Valadez V (1998) AVPR2 variants and V2 vasopressin receptor function in nephrogenic diabetes insipidus. Kidney Int 54: 1909–1922.
- 26. Wildin RS, Antush MJ, Bennett RL, Schoof JM, Scott CR (1994) Heterogeneous AVPR2 gene mutations in congenital nephrogenic diabetes insipidus. Am J Hum Genet 55: 266–277.
- 27. Arthus MF, Lonergan M, Crumley MJ, Naumova AK, Morin D, et al. (2000) Report of 33 novel AVPR2 mutations and analysis of 117 families with X-linked nephrogenic diabetes insipidus. J Am Soc Nephrol 11: 1044–1054.
- 28. Seibold A, Brabet P, Rosenthal W, Birnbaumer M (1992) Structure and chromosomal localization of the human antidiuretic hormone receptor gene. Am J Hum Genet 51: 1078–1083.
- 29. Murasawa S, Matsubara H, Kijima K, Maruyama K, Mori Y, et al. (1995) Structure of the rat V1a vasopressin receptor gene and characterization of its promoter region and complete cDNA sequence of the 3′-end. J Biol Chem 270: 20042–20050.
- 30. Kubota Y, Kimura T, Hashimoto K, Tokugawa Y, Nobunaga K, et al. (1996) Structure and expression of the mouse oxytocin receptor gene. Mol Cell Endocrinol 124: 25–32.
- 31. Spanakis E, Milord E, Gragnoli C (2008) AVPR2 variants and mutations in nephrogenic diabetes insipidus: review and missense mutation significance. J Cell Physiol 217: 605–617.
- 32. Schulz A, Schoneberg T (2003) The structural evolution of a P2Y-like G-protein-coupled receptor. J Biol Chem 278: 35531–35541.
- 33. Staubert C, Tarnow P, Brumm H, Pitra C, Gudermann T, et al. (2007) Evolutionary aspects in evaluating mutations in the melanocortin 4 receptor. Endocrinology 148: 4642–4648.
- 34. Schoneberg T, Hermsdorf T, Engemaier E, Engel K, Liebscher I, et al. (2007) Structural and functional evolution of the P2Y(12)-like receptor group. Purinergic Signal 3: 255–268.
- 35. Schoneberg T, Yun J, Wenkert D, Wess J (1996) Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. Embo J 15: 1283–1291.
- 36. Pan Y, Wilson P, Gitschier J (1994) The effect of eight V2 vasopressin receptor mutations on stimulation of adenylyl cyclase and binding to vasopressin. J Biol Chem 269: 31933–31937.
- 37. Schulein R, Liebenhoff U, Muller H, Birnbaumer M, Rosenthal W (1996) Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site. Biochem J 313(Pt 2): 611–616.
- 38. Schulein R, Hermosilla R, Oksche A, Dehe M, Wiesner B, et al. (1998) A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells. Mol Pharmacol 54: 525–535.
- 39. Krause G, Hermosilla R, Oksche A, Rutz C, Rosenthal W, et al. (2000) Molecular and conformational features of a transport-relevant domain in the C-terminal tail of the vasopressin V(2) receptor. Mol Pharmacol 57: 232–242.
- 40. Rovati GE, Capra V, Neubig RR (2007) The highly conserved DRY motif of class A G protein-coupled receptors: beyond the ground state. Mol Pharmacol 71: 959–964.
- 41. Wu S, Birnbaumer M, Guan Z (2008) Phosphorylation analysis of G protein-coupled receptor by mass spectrometry: identification of a phosphorylation site in V2 vasopressin receptor. Anal Chem 80: 6034–6037.
- 42. Innamorati G, Sadeghi H, Eberle AN, Birnbaumer M (1997) Phosphorylation of the V2 vasopressin receptor. J Biol Chem 272: 2486–2492.
- 43. Innamorati G, Sadeghi HM, Tran NT, Birnbaumer M (1998) A serine cluster prevents recycling of the V2 vasopressin receptor. Proc Natl Acad Sci U S A 95: 2222–2226.
- 44. Bichet DG (2008) Vasopressin receptor mutations in nephrogenic diabetes insipidus. Semin Nephrol 28: 245–251.
- 45. Ortiz RM (2001) Osmoregulation in marine mammals. J Exp Biol 204: 1831–1844.
- 46. King JM, Bradshaw SD (2008) Comparative water metabolism of Barrow Island macropodid marsupials: hormonal versus behavioural-dependent mechanisms of body water conservation. Gen Comp Endocrinol 155: 378–385.
- 47. Bradshaw SD, Morris KD, Bradshaw FJ (2001) Water and electrolyte homeostasis and kidney function of desert-dwelling marsupial wallabies in Western Australia. J Comp Physiol [B] 171: 22–32.
- 48. Dawson TJ, McTavish KJ, Munn AJ, Holloway J (2006) Water use and the thermoregulatory behaviour of kangaroos in arid regions: insights into the colonisation of arid rangelands in Australia by the Eastern Grey Kangaroo (Macropus giganteus). J Comp Physiol [B] 176: 45–53.
- 49. Brownfield MS, Wunder BA (1976) Relative medullary area: a new structural index for estimating urinary concentrating capacity of mammals. Comp Biochem Physiol A Comp Physiol 55: 69–75.
- 50. Schmidt-Nielsen B, O'Dell R (1961) Structure and concentrating mechanism in the mammalian kidney. Am J Physiol 200: 1119–1124.
- 51. Beuchat CA (1990) Body size, medullary thickness, and urine concentrating ability in mammals. Am J Physiol 258: R298–308.
- 52. Adan RA, Kas MJ (2003) Inverse agonism gains weight. Trends Pharmacol Sci 24: 315–321.
- 53. Kleinau G, Jaeschke H, Mueller S, Worth CL, Paschke R, et al. (2008) Molecular and structural effects of inverse agonistic mutations on signaling of the thyrotropin receptor–a basally active GPCR. Cell Mol Life Sci 65: 3664–3676.
- 54. Adan RA (2006) Constitutive receptor activity series: endogenous inverse agonists and constitutive receptor activity in the melanocortin system. Trends Pharmacol Sci 27: 183–186.
- 55. Ballesteros JA, Weinstein H (1995) Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci 25: 366–428.
- 56. Sangkuhl K, Rompler H, Busch W, Karges B, Schoneberg T (2005) Nephrogenic diabetes insipidus caused by mutation of Tyr205: a key residue of V2 vasopressin receptor function. Hum Mutat 25: 505.
- 57. Schoneberg T, Schulz A, Biebermann H, Gruters A, Grimm T, et al. (1998) V2 vasopressin receptor dysfunction in nephrogenic diabetes insipidus caused by different molecular mechanisms. Hum Mutat 12: 196–205.