sign in sign up
<<
Home , Desmopressin, Diabetes insipidus, Vasopressin analogue

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector

Kidney International, Vol. 54 (1998), pp. 1909–1922

AVPR2 variants and V2 function in nephrogenic insipidus

ROBERT S. WILDIN,DAVID E. COGDELL, and VICTORIA VALADEZ

Department of Molecular and Medical , Oregon Health Sciences University, Portland, Oregon, USA

AVPR2 variants and V2 function in neph- Other AVPR2 mutations with milder effects on receptor function rogenic . probably exist, but may not be expressed clinically as typical NDI. Background. The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/ receptor subfam- ily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the Congenital nephrogenic diabetes insipidus (NDI) is an functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of inborn error of water caused by insensitivity of naturally occurring AVPR2 mutations constitutes a model system the distal renal tubule and collecting duct to the effects of for studying the structure-function relationship of G protein- the pituitary-derived vasopressin (AVP). coupled receptors. This analysis can be aided by examining amino Two genetic forms of NDI are known. The more common acid sequence variation and conservation among evolutionarily form results from mutations in the type 2 receptor for AVP disparate members of the subfamily. Methods. Twenty-five new NDI patients were evaluated by (V2 receptor), a G protein-coupled receptor, that inhibit its DNA sequencing for mutations in AVPR2. Receptors encoded by capacity to respond to increases in serum AVP [1–5]. Since eighteen NDI alleles were tested for physiologic signaling activity the V2 receptor gene, AVPR2, resides in chromosome in response to varying concentrations of arginine vasopressin band Xq28, this form of the is inherited in an (AVP) in a sensitive cell culture assay. Seventeen amino acid X-linked fashion. Like other X-linked disorders with his- sequences from the vasopressin/ subfamily were torically reduced probability of reproduction in the affected aligned and conserved residues were identified and correlated with the locations of NDI associated variations. males, new mutations are frequent. Results. Twenty-four variant alleles were found among the 25 In other NDI cases, a transmembrane water channel new patients. Thirteen had no prior family history of expressed protein, -2, is defective [6–11]. The encoding NDI. All 18 of the NDI-associated AVPR2 alleles tested for gene, AQP2, resides on chromosome 12, so its inheritance function demonstrated diminished response to stimulation with is autosomal. The majority of cases thus far described have AVP. Twelve failed to respond at all, whereas six signaled only at occurred in families with known consanguinity. Proper high AVP concentrations. Evolutionarily conserved residues clus- tered in the transmembrane domains and in the first and second genetic counseling of NDI patients and their families, extracellular loops, and NDI-associated missense mutations ap- especially those without an informative family history, peared mostly in the conserved domains. requires that the two forms be distinguished. Furthermore, Conclusions. Sporadic cases are frequent and they usually as the science of this disorder progresses, new treatments represent the X-linked rather than the autosomal form of NDI. aimed at one or the other form will be devised, and having Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to a clear picture of the abnormal biology in an individual case retain partial signaling in vitro, whereas those that introduce a will be imperative. charged residue in a transmembrane domain are inactive. The In addition to its clinical relevance, the study of varia- minimal partial signaling observed in cultured cells is unlikely to tions that affect the function of the V2 receptor has correlate with clinically significant concentrating ability. scientific value. When combined with other research, it may help decypher how the receptor’s structure defines its function, and may even provide insight into the function of Key words: congenital nephrogenic diabetes insipidus, arginine vasopres- the large numbers of G protein-coupled receptors. sin, genetic testing, protein structure, gene mutations, water homeostasis, These receptors comprise a diverse and ancient super- signal transduction, aquaporin-2, G protein-coupled receptors. family of transmembrane signaling proteins. All couple to a Received for publication April 9, 1998 less extensive collection of heterotrimeric G proteins and and in revised form July 2, 1998 share the apparently invariable characteristic of seven Accepted for publication July 6, 1998 hydrophobic transmembrane domains. However, since few © 1998 by the International Society of amino acid residues have been completely conserved in

1909 1910 Wildin et al: V2 receptor defects in NDI evolution across the entire superfamily, comparisons of Amplification products were purified by gel electro- primary amino acid sequence imply little about the funda- phoresis in 1% agarose (FMC corporation) in TAE buffer mental structure-function relationship of these membrane [14] followed by extraction with the Wizard PCR prep kit proteins [12]. Unfortunately, the complete tertiary struc- (Promega, Madison WI, USA). 400 ng of product was ture of a member of the superfamily has not been deter- subjected to cycle sequencing using the PST-RR Dye mined experimentally. Terminator mix (#402124) supplied by Perkin-Elmer. Se- It is possible, however, to infer functional domains based quencing primers (5Ј-Ͼ3Ј) were BW106 (CCT GTG TCG on the abnormal behavior of receptors with modified amino GGC CGT GAA GTA), BW102 (GTG GCC AAG ACT acid sequences. One aspect of this approach involves GTG AGG ATG), BW107(CCA ATG AAG ACG TGT analyzing receptors with mutations that reduce or eliminate ATG GGT), BW213 (CCA GGG TAG GTG CCA CGA the ability to transduce a signal. The V2 vasopressin ACA) and occasionally BW100 (TGC TCC TCA GGC receptor is unique among the mammalian G protein- AGA GGC TGA). coupled receptors in that many functionally impaired ver- Sequence electropherograms obtained on a model 373 or sions are available from patients with NDI. 377 automated sequencer (Applied Biosystems) were visu- We previously reported eleven AVPR2 gene mutations ally confirmed using Factura software (Applied Biosys- associated with NDI in humans [13]. We now report 24 tems) and then compared with wild-type sequence using additional mutations and present evidence of reduced or the GeneWorks sequencing project software (Oxford Mo- eliminated function of the encoded receptors for 18 of lecular). them. This confirms their causal relationship to the disor- DNA sequence numbering is according to GenBank der, and sets the stage for an experimental analysis of accession number U04357, the genomic AVPR2 sequence structure and function at the molecular level. [13]. Note that the original sequence has one error, a missing T at position 144 in intron A. The GenBank entry METHODS has been corrected.

Patients Cloning and expressing mutant genes Blood samples were obtained with informed consent The entire AVPR2 gene was amplified by PCR from from patients with a clinical diagnosis of Nephrogenic genomic DNA using Pfu (or, rarely, Taq) polymerase and Diabetes Insipidus. All reportedly had evidence of abnor- the manufacturer’s buffer (Stratagene or Perkin-Elmer). mally low responsiveness to injected vasopressin or desmo- The amplification primers were BW216 (aag gta cCT GAG pressin (a selective for the V2 recep- TCC GCA CAT CAC CTC C) and BW217 (tgg agc tcA tor), or they had a history of an affected male relative with GGG ATT AGA AAG GCG GAG ACA). Bases in lower abnormally low responsiveness. case were added to create KpnI and SacI restriction sites. The amplification parameters were as above, except that Genomic DNA extraction four 25 ␮l reactions with 125 ng of DNA template each were employed. Genomic DNA was extracted from 3 to 7 ml of periph- Amplification products were gel purified and extracted as eral blood anticoagulated with EDTA or with ACD using above, digested with restriction endonucleases SacI and either PureGene kit (Gentra Systems, Research Triangle KpnI (Pharmacia), and subcloned into the plasmid vector Park, NC, USA) or an organic extraction protocol em- pCD/PS [15]. ployed by the DNA Diagnostic Laboratory at Oregon Health Sciences University. cAMP response assay JEG3 human choriocarcinoma cells (American Type Avpr2 amplification and direct sequencing Culture Collection) were plated in 24-well dishes at 4.8 ϫ The entire AVPR2 gene was amplified using the poly- 104 or 2.4 ϫ 104 cells/well one to two days, respectively, merase chain reaction (PCR) from 125 ng of genomic DNA prior to transfection and cultured in Dulbecco’s modified using the XL-PCR kit (Applied Biosystems) and the fol- Eagle medium (DMEM) plus 10% Fetal Clone III (Hy- lowing primers (5Ј-Ͼ3Ј) in the PTC-200 DNA Engine (MJ clone) supplemented with penicillin/streptomycin (10 U/ml, Research): BW204 (TGG AAT TCG GCT TTG CTC CTC Sigma), L-glutamine (2 mM, Life Technologies) and 0.1 mM AGG CAG AGG CTG A) and BW205 (CAG AAT TCG non-essential amino acids (Life Technologies). GCT TAG GGA TTA GAA AGG CGG AGA CA). Cells were transfected using phosphate precipi- The amplification parameters were: hot start at 94°C, tation [16] when cell density reached 70% confluency. cycle 94°C for 30 seconds, and 66°C for five minutes, fifty Culture media was replaced with 0.25 ml transfection times, followed by 72°C for 10 minutes, and a 10°C soak. media [DMEM plus 0.5% bovine serum albumen, fraction Two reactions of 50 ␮l each under 25 ␮l of oil were V (Sigma), supplemented as above] and equilibrated. Each performed. well then received 10 ng of plasmid expressing the wild-type Wildin et al: V2 receptor defects in NDI 1911

or mutant receptor, 15 ng of the alpha-inhibin-luciferase variants, to produce functional receptors when expressed in reporter plasmid [17], which is cAMP responsive, and 40 ng cell culture. A significant variation from normal in the of a constitutively active ␤-galactosidase reporter plasmid ability to transduce signal in vitro suggests that an AVPR2 [18] to control for transfection efficiency, with pCD/PS to a variation is actually a disease-causing mutation. ␮ ␮ total of 250 g plasmid as 25 l of CaPO4 precipitate suspension. Twenty-four hours after transfection, media Nephrogenic diabetes insipidus subjects was replaced with transfection media containing no addi- AVPR2 alleles from 25 unrelated subjects with a clinical tions, AVP (Sigma) at varying concentrations, DDAVP diagnosis of NDI were studied (Table 1). Blood samples (Sigma) at 10 nM, or forskolin (Sigma) in ethanol at 10 ␮M. were obtained with informed consent by the subjects’ Each condition was performed in triplicate. treating and, in most cases, mailed to the authors Two and a half hours after stimulation, wells were from distant locations. Twenty-three of the subjects were aspirated and the cells were lysed in 100 ␮l/well of 1 X males, while two were females whose NDI symptoms Reporter Lysis Buffer (Promega), shaken for 10 minutes at required medical attention. All these patients had clinical room temperature, then frozen at Ϫ80°C. The thawed evidence of a defect in urine concentrating ability that was lysates were harvested and centrifuged at 3,000 ϫ gat4°C resistant to parenteral infusion of vasopressin, or desmo- for 10 minutes. pressin (DDAVP), or such evidence existed for a first or Luciferase activity was measured in 20 ␮l of lysate second degree affected male relative. The available data supernate in a Berthold 953 Autolumat luminometer by from clinical records reporting the diagnostic studies for injecting 100 ␮l of Luciferase assay reagent (Promega) and patients whose mutations were analyzed for function are integrating over 15 seconds. ␤-galactosidase activity was shown in Table 2. measured colorometrically at 410 nm using a previously There was no prior affected male in the families of 13 of described assay [19]. the 23 patients for whom family history was available. We Luciferase activity was corrected for transfection effi- defined these as sporadic cases because they occurred ciency by dividing by the associated ␤-galactosidase activity. without prior knowledge of clinical disease in the family. Standard deviations (SDs) were calculated on triplicate Genetic counseling is challenging in such cases because experiments. Data analysis and graph preparation made they may, in reality, represent inherited X-linked muta- use of Microsoft Excel and CricketGraph software. tions, new mutations in AVPR2 not present in the patient’s parents, autosomal recessive AQP2 mutations, cases of Sequence alignments non-genetic NDI, or other disorders having NDI as a Seventeen amino acid sequences from various species feature. In four of the 13 sporadic cases in our cohort, the representing V2Rs, oxytocin receptors, V1a and V1b re- patient’s mother, and sometimes other female relatives, ceptors, and the related amphibian bladder mesotocin had subclinical complaints of and . The receptor and teleost isotocin receptor were aligned using age at definitive diagnosis ranged from two weeks to eight the classical algorithm and manually adjusted in Gene- years, with the majority diagnosed within the first four Works software (Oxford Molecular). The sequence acces- months of life. The racial backgrounds of the subjects sion numbers are: Swissprot: P30518 (V2R_HUMAN), included Caucasian/northern European (N ϭ 13), East Q00788 (V2R_RATNORV), P48044 (V2R_BOVIN), Indian (N ϭ 1), African (N ϭ 1), Okinawa (N ϭ 1), Latino P32307 (V2R_PIG), P32306 (OXYR_PIG), Q28756 (N ϭ 2), and unknown (N ϭ 7). (OXYR_SHEEP), P30559 (OXYR_HUMAN), P70536 (OXYR_RAT), P37288 (V1AR_HUMAN), P47901 AVPR2 variations in nephrogenic diabetes insipidus (V1BR_HUMAN), P48043 (V1AR_SHEEP); PIR: I50130 patients (ICTCN_WSUCKR); GenBank: D86599 (OXYR- The genomic DNA sequence of AVPR2 was completely _MUSSp), U82440 (OXYR_MACAC), X93313 (MSTC- determined for each patient by direct sequencing of PCR N_TOAD); EMBL: Z11932 (V2R_RATRAT); and Wildin product. Differences from the published reference se- et al (unpublished data): V2R_MOUSE. Statistical calcu- quence were called “variations,” and the alleles in which lations of significance were performed by the ⌾2 method they were found termed “variant,” because their effect on using the Yates correction [20]. the function of the encoded V2 receptor was not immedi- ately known. Variants whose function is indistinguishable RESULTS from the reference wild-type allele are properly termed We previously reported eleven NDI patients with se- polymorphisms, while those associated with measurable quence variations in AVPR2 [13]. In this study, we wished differences may ultimately be denoted mutant. Twenty-four to confirm the high frequency of novel AVPR2 variations different variant alleles of AVPR2 were present among the by studying more NDI patients. In addition, we assessed 25 study subjects (Table 1). Two patients who were not their causal relationship to the disease by testing the ability known to be related harbored the same variation. Several of the altered alleles, particularly those predicting missense had variations reported in previous NDI studies (Table 1, 1912 Wildin et al: V2 receptor defects in NDI

Table 1. AVPR2 NDI alleles (this study)

Ethnicity Positive past family Age at AVPR2 Previous ID Gender (extraction) history diagnosis variations Predicted Consequences to V2R observation 96–10 M African 2 maternal uncles 4 months A214G, Silent in intron A, R139S, C847A, frameshift after R139 and TER 850delG after 21 more codons 972121 M East Indian Unknown 6 weeks G467A, G12E, [40] (G12E ϩ T1493C C319R L292P) 96–05 M US Latino None 1 month A595G N55D 96–03 M US Caucasian None ϩ mother 4 months ϩ G675C L81F ϩ 3–124 symptomatic 8 years 96–09 M Canadian None 22 months A707G Q92R Caucasian 93–362 M Unknown Unknown Unknown 766delTG Frameshift after L111 and TER after 78 more condons 93–397 M US Caucasian 2 maternal uncles Unknown C769T R113W [36, 37, 39] 96–06 M GB Unknown Maternal 2nd cousin 3 months G796C G122R 93–530 M US Caucasian None 2 months 838del40 Deletion of 13 residues with frameshift after L135 and TER after 12 codons 97–01 M US Hawaiian Extensive 3 months 874insG Y148X (Okinawa) 96–01 M Unknown Maternal half-brother 2 weeks C932T S167L [13, 36, 37, 42] 96–04 M US Caucasian Extensive 3 weeks C973T, R181C, (R181C only [36, (N. European) G1075A V215M 37] or with deletion [3]) 972888 M US Caucasian Mother symptomatic 4 months 1044insC Frameshift after T204, TER after 53 more codons 96–02 M US Unknown Mother, 2 sisters 3 weeks T1088C L219P [43] symptomatic 97–22 M US Unknown Extensive 2 weeks 1216delG Frameshift after H261, TER after [38] (base not 8 more codons mentioned) 97–11 M US Latino None 5 months T1247A M272K 972462 F US Unknown None 4 years 1267del3 ⌬V279 [41, 44] 97–13 M US Caucasian None 6 months G1311A W293X [36] (different base, same consequence) 971838 M US Caucasian 2 maternal uncles and 16 weeks G1343A Disrupts 5Ј spice signal for intron (Ireland) one male cousin B (AAGgtggg3 AAGatggg) 971819 M US Caucasian Extensive 2 weeks T1473A L312X [36, 45] 97–09 M US Caucasian Mother symptomatic 4 months C1483A S315R (English) 95–13 M US Unknown None 9 months C1489A N317K 96–08 M GB Caucasian None 10 months C1498A, T320silent, A1499T N321Y 97–28 F US Caucasian Father and 2 paternal 1 year 1507delG W323X (German) half-uncles

column 8), including one (S167L) from our prior study [13]. terminal peptides. The allele (96-01) harboring one such The two female subjects were each heterozygous for small deletion also contained a silent change in the first intron, as deletions that were easily detected in the sequence electro- well as an amino acid substitution in the codon just pherograms. One additional patient with diabetes insipidus preceding the deletion. had no detectable variations in AVPR2. He was excluded Two single-base substitutions (G1311A, T1473A), one from the cohort reported here because reduced responsive- single-base deletion (1507delG, from a female subject), and ness to vasopressin had not been documented. one single-base insertion (874insG) create premature ter- In most cases, only a single DNA sequence variation was mination (nonsense) codons. In another allele, a substitu- found in each mutant allele. Four alleles had two or more tion (G1343A) in the critical ϩ1 position of the splice variations (the common silent polymorphism in the codon donor for the second intron likely results in aberrant for leucine at position 309 was not counted as a variation). splicing and an altered messenger RNA. The variant allele All variant alleles predicted a change in the amino acid from a second female subject (1267del3) revealed an sequence of the AVPR2 polypeptide. Four deletions in-frame, three base-pair deletion predicting the loss of (850delG, 766delTG, 838del40, and 1216delG) and one Valine 278 or 279, but an otherwise normal receptor insertion (1044insC) are predicted to cause frameshifts protein. The remaining alleles harbored one or two single with early chain termination and alteration of the carboxy- base-pair substitutions that predict amino acid substitutions Wildin et al: V2 receptor defects in NDI 1913

Table 2. Clinical data from subjects whose variant V2 receptors were tested in Figure 1

Amino acid ID change Urine concentration defect 96–08 N321Y 4 hour water deprivation test: maximum serum/urine Osma 305/30, followed by 0.4 ␮g intramuscular DDAVP 3 serum/urine Osm 311/53, with no subsequent response to high dose DDAVP. Results confirmed at 4 years of age following trial of intranasal DDAVP up to 20 ␮g/dose. 97–13 W293X Presenting serum 158 mEq/liter. “He did not respond to of ADH” 93–397 R113W No testing records; family history of X-linked disease 3–124 L81F Short stature, FTTb; Pitressin challenge - urine Osm, “responded somewhat” 96–03 L81F Testing records not available; sporadic 96–02 L219P Spontaneous serum/urine Osm 323/19; after 0.2 ␮g subcutaneous DDAVP minimum serum Osm 324, maximum urine Osm 106 96–06 G122R No testing records; family history of X-linked disease 88–082 Y205C No testing records; family history of X-linked disease 87–151 W323R No testing records; family history of X-linked disease 93–530 D136fs No testing records; family history of X-linked disease; hospitalized 14 times by age 2.5 years for hypernatremic 93–169 S167L Spontaneous serum/urine Osm 360/179; “No response” to 0.05, 0.1 or 0.2 cc intranasal DDAVP, maximal urine Osm 196 96–01 S167L No testing records; maternal half-brother is affected 92–233 P95L No testing records; adopted 95–13 N317K Spontaneous serum/urine Osm 317/131; urine before/after subcutaneous DDAVP: output ϭ 5.5/4.6 cc/kg/hr, maximum Osm 109/108 96–05 N55D Presenting serum sodium 156 mEq/liter; vasopressin challenge: 0.115 ␮gupto5␮g gave “no response” 93–141 L53R No testing records; has younger affected sister 97–09 S315R Water deprivation test: dehydrated to 96% body weight, serum/urine Osm 330/173; DDAVP challenge 20 ␮g intranasal 3 “no response”, then 2 ␮g intravenous 3 “no response.” ADH level Ͼ 40 pg/ml 97–11 M272K Presenting serum sodium 161 mEq/liter serum/urine Osm 352/111, later 367/153; no decrease in urine output after 0.01 ␮g intranasal DDAVP or after 1 Unit subcutaneous Pitressin, urine specific gravity remained below 1.005 96–04 R181C ϩ Spontaneous serum/urine Osm 327/88; “he did not respond to [a vasopressin stimulation test] in the hospital” V215M 972121 G12E ϩ Adopted. Presenting serum sodium 161 mEq/liter serum/urine Osm 335/126, later 323/151; urine Osm 142 (unchanged) after C319R 5 ␮g intranasal DDAVP, maximum of 158 one hour after parenteral aqueous pitressin a Osm is osmolality, mOsm/kg b FTT, failure to thrive

(missense) only. Two alleles had two changes each: 972121 cloned the subject’s AVPR2 gene into an expression plas- with G12E and C319R substitutions, and 96-04 with R181C mid, transfected it into a V2 receptor-negative human and V215M substitutions. If a conservative amino acid tumor cell line, and tested the ability of the transfected cells substitution is defined as one resulting in no net electrical to respond to AVP via the experimentally expressed recep- charge and no change in the polarity of a neutral residue, tors. Since the subjects’ genes were cloned using PCR, then four of the non-polymorphic missense variations ob- which can result in the introduction of base change arti- served predict conservative changes (L81F, V215M, L219P, facts, we used a high-fidelity polymerase. The sequence of N321Y) and ten predict non-conservative changes. each cloned insert was then determined to verify its identity Functional characteristics of AVPR2 mutations to that obtained directly from the subject’s independently amplified PCR product. We chose to test the missense Theoretically, some AVPR2 sequence variations may not variations, because they are much more likely to represent cause any change in the function of the encoded receptor, masquerading polymorphisms than those changes predict- although they are found in patients with a clinical diagnosis ing protein truncation. We included two from the latter of NDI. They represent polymorphisms, or normal varia- category (one nonsense and one frameshift) in our selec- tions that may be found in individuals not affected with NDI, or even in an individual affected with NDI that is due tion as negative controls. to mutations in another gene, like AQP-2. The transfected cells’ ability to respond to AVP via the From a clinical genetics perspective it is therefore valu- V2 receptors was measured in an assay previously used to able to establish whether or not an AVPR2 gene sequence assess G protein signaling pathways coupled to the adren- variation from an NDI patient indeed results in a loss of ergic and muscarinic receptors [21]. A plasmid bearing a receptor function. This is particularly important in cases of cAMP-responsive promoter driving a luciferase reporter NDI without a strong X-linked family history. Defining the gene is co-transfected into the cells with the V2 receptor functional capacity of variant receptors is likewise essential expression plasmid. Physiologic increases in cAMP due to for the scientific analysis of the structure-function relation- agonist-induced activation of the expressed V2R are re- ship of the seven-transmembrane-domain protein. flected in increased luciferase transcription, translation, To test the function of the putative mutant receptors, we and enzymatic activity. This occurs because elevations of 1914 Wildin et al: V2 receptor defects in NDI cAMP activate (PKA), which phosphory- V2 receptor-expressing cells with 10 ␮M forskolin demon- lates a transcription factor (CREB), in turn resulting in the strated that the reporter system was intact in each experi- activation of transcription at gene promoters bearing ment, and that the level of maximal stimulation by the CREB binding sites, such as the ␣-inhibin promoter in the wild-type receptor was similar to that induced by receptor- luciferase plasmid. Since activation of PKA is felt to be independent mechanisms (Fig. 1, right side). involved in the fusion of aquaporin-2-laden vesicles with Increasing the amount of reporter plasmid transfected the apical membrane of the collecting duct epithelium in increased the overall level of luciferase activity, but it did response to AVP stimulation, this downstream readout not change the concentration at which the maximal lucif- mimics the events occurring with activation of the V2 erase activity was first reached (not shown). This suggests receptor in vivo. Furthermore, the wild-type receptor in this that the rate-limiting step responsible for the plateau in the assay responds to concentrations of AVP that cause water signaling response at AVP concentrations above 0.032 nM conservation in normal individuals (Fig. 1). in wild-type resides in the signaling pathway upstream of Because the transfection efficiency may vary between luciferase transcription activation. Control cells that were different wells in the same experiment, a third plasmid with not transfected did not have detectable luciferase activity. a constitutively active ␤-galactosidase gene is also included Cells receiving the luciferase and ␤-galactosidase reporter in the transfection. Luciferase activity in the cell lysates is plasmids plus the pCD/PS expression plasmid without a V2 then divided by the ␤-galactosidase activity to correct for receptor gene insert had basal luciferase activity similar to transfection efficiency. The assay is extremely sensitive, it the fully transfected, but unstimulated cells (not shown). reflects a physiologic signaling cascade, and it does not The experimental results obtained from this assay were depend on artificial elevation of cAMP levels by pharma- reproducible on different occasions. cological inhibition of phosphodiesterase. Eighteen variant AVPR2 alleles from NDI patients, two Amino acid conservation in related receptors identical wild-type alleles from different subjects (wt and Conservation of amino acid residues over long periods of wt2), and one allele with three mutations generated by evolution is an acceptable predictor of structural or func- PCR error (M1V ϩ G185S ϩ I221T) were tested in this tional importance. Members of the vasopressin/oxytocin fashion (Fig. 1). All NDI alleles demonstrated a substan- receptor subfamily are found broadly in vertebrate species, tially decreased ability of the altered receptor to activate including amphibians and fishes. Comparing their se- the luciferase reporter in response to AVP. Twelve clearly quences provides a means to query which amino acid had no response to AVP at concentrations as high as 320 residues may be most important in the overall function of 5 nM,10-fold higher than the lowest concentration tested, the receptors. Since they all bind quite similar natural where the wild-type receptor had clearly detectable activity. ligands, their comparison over wide evolutionary distances Six of the NDI alleles (N321Y, R113W, L81F, P95L, is more meaningful than a comparison with other G N317K, and R181C/V215M) showed partial or complete protein-coupled receptors that have very different natural responses at AVP concentrations from 1.5 logs to four logs agonists, and thus have had different evolutionary con- higher than required for the wild-type. The second wild- straints. type allele, wt2, the normal allele from a heterozygous We extracted from the public databases protein se- female, responded to AVP in a manner identical to the quences whose level of similarity to the human V2R was first. significant, as defined by the XrefDB algorithm [22], and The allele bearing the three PCR-generated mutations aligned their amino acid sequences. Seventeen sequences (M1V, G185S, and I221T) demonstrated nearly normal were used (Fig. 2). They included type 2 vasopressin function in this assay. The level of signal obtained at the receptors from six mammals, oxytocin receptors from six three lowest AVP concentrations tested was significantly mammals, type 1a vasopressin receptors (V1a) from two lower for this allele than for wild-type, yet it was measur- mammals, type 1b vasopressin receptor from human (V1b), able and therefore greater than those of the patient derived and two more distant relatives: the isotocin receptor from alleles. the White Suckerfish and the mesotocin receptor from the In order to confirm that the luciferase activity observed Black Toad. was due to activation of the V2 receptor derived from the Eighty-five residues, excluding the initiating methionine, transfected genes, and to search for major mutation-related were completely conserved among the seventeen receptors, changes in agonist discrimination, the cells were stimulated suggesting that they may play a role in ligand binding, in in parallel with 10 nM (dDAVP), a V2- establishing the necessary secondary or tertiary structure, selective agonist (Fig. 1, right side). In all cases, the level of or in transmitting an activation signal to G proteins. The corrected luciferase activity stimulated by 10 nM dDAVP preponderance of wholly conserved residues lies in the was similar to that induced by 10 nM AVP. transmembrane domains and in the first and second extra- The diterpene forskolin stimulates adenylyl cyclase inde- cellular loops. The palmitoylation site (CC) and the more pendent of G protein signaling. Parallel stimulation of the broadly conserved domain in the seventh transmembrane Wildin et al: V2 receptor defects in NDI 1915

Fig. 1. V2 receptor arginine vasopressin (AVP) dose response. Each pair of panels represents a single set of triplicate experiments, and each data point and associated bar represent the mean and SD, respectively. The horizontal axis of the left panels is marked in half-log increments of AVP concentration. The vertical axis is expressed as Luciferase activity (in arbitrary light units) divided by ␤-galactosidase activity (in arbitrary absorbance units). The data are normalized to wild-type activity to facilitate comparison between panels. The baseline wild-type activity is defined as zero and the maximum mean wild-type activity for each panel is defined as 100%. Variants shown here but not listed in Table 1 were previously reported [13]. 1916 Wildin et al: V2 receptor defects in NDI

Fig. 1. Continued Wildin et al: V2 receptor defects in NDI 1917 segment are also among the completely conserved sites. In carrier of the disease allele is two thirds. It is reasonable to other G protein receptors, the cytoplasmic loops are exper- assume that the likelihood of reproduction in males with imentally implicated in receptor:G protein interactions NDI was limited in past generations, and may still be in [12]. Since members of this collection couple to different G medically under-served regions. protein subsets, it is not surprising that residues in the Invoking Haldane’s Indirect Method, this suggests that at cytoplasmic loops are commonly different. Close inspection least two thirds of mothers of sporadic NDI cases may be reveals a high level of conservation in some cytoplasmic carriers [25]. We conclude that mothers of sporadic males domains among receptors that signal via a similar G protein with NDI due to a mutation in AVPR2 are at substantial subset. risk of being carriers of a disease-causing allele, and carrier Inspection of Figure 2 also suggests that completely testing should be made available. Successful testing has conserved residues are disproportionately represented been done on an experimental basis [13, 26]. among the missense mutations associated with NDI. The 85 Equally important, locus heterogeneity for NDI clearly completely conserved residues are mutated 24 times (28%), exists in the form of autosomal mutations in aquaporin-2 whereas the remaining 286 residues not completely con- (AQP2). Thus, these sporadic patients cannot be assumed served were altered only 24 times (8.4%). The association is to harbor NDI-causing AVPR2 mutations, and any AVPR2 statistically significant (⌾2 with Yates correction ϭ 21.2, sequence variations found in them, especially those predict- p Ͻ 0.001). ing a missense only, must initially be suspect. This is particularly important if the presence of the mutation is to DISCUSSION be used as evidence of X-linked inheritance in genetic Genetic variation causes human disease when it changes counseling. Furthermore, the functional incapacity of the the expression of a gene or the function of its protein variant molecule must be clearly established if NDI-asso- product. New variations in the AVPR2 gene encoding the ciated variations are to be used to draw inference about the V2 vasopressin receptor are thus strongly implicated in the structure-function relationship of the V2 receptor. causation of congenital nephrogenic diabetes insipidus. All patients in this study demonstrated variations in the Nevertheless, variations without functional significance, or AVPR2 gene. The nonsense and frameshift changes are polymorphisms, have been reported. Identifying variations reasonably assumed to be mutations that eliminate recep- in AVPR2 in NDI patients and confirming their functional tor function; this was confirmed by testing one of each (Fig. significance are important for both accurate genetic coun- 1; D136fs and W293X). All but one of the missense seling and for advancing scientific understanding of G variations were tested and were shown to affect the func- protein-coupled receptors. tion of the V2 receptor (Q92R was not tested). Therefore, In this report, we describe a large cohort of independent- it is highly unlikely that any of these patients have NDI ly-ascertained NDI patients, including two females, each of solely as a result of defects in aquaporin-2, the action of whom has one or more genetic sequence variation in which is downstream of receptor signaling. This supports a AVPR2. Fifteen of the alleles have not been previously conclusion that the incidence of autosomal recessive NDI is reported. Altogether, over 80 different variant AVPR2 quite low in the sampled population relative to X-linked alleles have been associated with NDI. The large number of NDI, even when families with a history of NDI in prior novel mutations is consistent with an X-linked recessive generations are excluded. On the other hand, when con- disorder with reduced genetic fitness, and it presents a sanguinity among the parents is present, or when the challenge for clinical DNA diagnosis. proband is a girl, mutations in AQP-2 should be more strongly considered. Vargas-Pousou et al found ten of Sporadic cases twelve families with NDI to have AVPR2 variations; the Between this study and our previous report [13], 52% of other two had autosomal recessive aquaporin-2 defects the subjects represented sporadic cases, each with a novel [11]. In one of the autosomal recessive cases the parents AVPR2 variation. This diversity is further evidence against were first cousins, and in the other case the proband was a the once widely accepted, and now disproved, “Hopewell girl. Females who are heterozygous for an inactivating hypothesis” that all NDI patients in North America derived AVPR2 allele may show clinical disease as a result of from a single founder family [23, 24]. skewed X-inactivation [27, 28]. Since each of these sporadic cases was shown to have a mutation in AVPR2, it is possible that their mothers, and Signaling capacity among variant receptors potentially other female relatives, are unwitting carriers of NDI. Such carriers have a recurrence risk of 50% for NDI Non-functional alleles. An allele with a frame shift at in subsequent male births. residue 136 and one with a nonsense mutation at residue For X-linked disorders with a genetic fitness of zero in 293 gave no response in our sensitive assay of receptor males, like Duchenne muscular dystrophy, the proportion function, as expected. The majority of the missense muta- of sporadic cases where the mother of the patient is a tions also failed to signal when stimulated with AVP. The 1918 Wildin et al: V2 receptor defects in NDI Wildin et al: V2 receptor defects in NDI 1919

Fig. 2. Amino acid alignment of V2 receptor-like sequences. Putative AVP and dDAVP, albeit at substantially higher concentra- transmembrane segments are underlined. Completely conserved residues are noted below the alignment. Known non-polymorphic missense varia- tions of ligand. The L81F mutation was detected in each of tions associated with NDI are identified above the alignment. Variations two geographically separated families, but this does not from this paper are in bold, while previously reported ones are in plain necessarily indicate a recurrent mutation. The first patient, font (L43P,G107E [11]; L44F, L62P, D85N, T204N,V206D [37]; L44P, S167T [46]; L53R, L83P, P95L, S167L, W323R [13]; R64W, V88M, 3-124, was described as having a partial response to vaso- R106C, C112R, S126F, A147V, W164S, A285P [36]; H90R [47]; R113W pressin challenge when he was diagnosed at age eight [48]; Y128S, R181C, P286R [3]; A132D, R137H [4]; R143P [49]; G185C, (Table 2). His two nephews with the same allele have R202C, Y205C [5]; T207N, L219P [43]; V277A, Y280C, L292P [39]; P286L [44]; L309P [50]; P322S, P322H [51]; L219R [28]). Missense reportedly had a variable disease course, suggesting that changes found cis to another missense variation are underlined. See partial receptor activity in vitro phenotype may correlate Methods for sequence names and accession numbers. with a milder clinical phenotype. Leucine is nearly invariant 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ at this position among more than 150 G protein-coupled receptors [29], being replaced only rarely with methionine or threonine. The hydrophobic nature of phenylalanine N55D variation alters a residue that is universally con- may be tolerated in this second transmembrane domain, served among more than 150 representative G protein- but the larger aromatic ring may displace the helix suffi- coupled receptors (http://mgddk1.niddk.nih.gov:8000/com- ciently to partially impair ligand binding or G protein- prehensive.html) [29], suggesting it is not only important coupling. for the vasopressin/oxytocin family, but is essential for all G The P95L mutation at the extracellular end of the second protein-coupled receptors. It also inserts a charged residue transmembrane domain showed a small response to very in the middle of a transmembrane domain. The L53R, high concentrations of AVP, concentrations much higher G122R, M272K, S315R, C319R, and W323R variations than one would expect to find in vivo. It seems unlikely that (and the untested Q92R) each place a charged residue in a this poor response is distinguishable from no response in transmembrane domain, and may thereby disrupt proper vivo. Since leucine is seen at this position in a number of assembly of the receptor. other G protein-coupled receptors (dopamine D4, melato- Substitution of serine by leucine at residue 167 also nin, some 5HT receptors, muscarinic receptors) [29], this eliminates signaling in our assay. Previously, Oksche et al change may not disrupt overall receptor structure, but showed that adenylate cyclase stimulation by the S167L rather alter part of an important ligand binding domain. variant is absent along with a total lack of cell surface AVP Indeed, Kojro et al showed that P95 is within a V2 receptor binding [9]. Immunoblot analysis indicated lack of post- segment that contacts AVP in photoaffinity labeling exper- translational processing and probably increased degrada- iments [32]. tion, supporting a conclusion that the nascent protein is not The function of the R113W mutation has been tested in properly targeted to the plasma membrane. The reason cell lines by Birnbaumber, Gilbert and Rosenthal [33]. why a non-polar substitution for this nucleophile in the They found a normal abundance of binding sites, but a middle of the fourth transmembrane domain prevents reduced kD of approximately 100 nM versus 5.3 nM for the receptor assembly remains unknown. wild-type receptor. The EC50 of adenylyl cyclase activity in The substitution of proline for leucine at position 219 in cell homogenates was 25- to 60-fold higher for the mutant the fifth transmembrane domain likely kinks an important receptor than for the wild-type. Our data, obtained in intact ␣ helix and may disrupt protein targeting as well, account- cells, demonstrate a shift in the concentration of AVP ing for its lack of activity. Lastly, cysteine in place of required for equivalent signaling of approximately 60-fold, tyrosine at position 205 in the C-terminal end of the second entirely consistent with the previous results. extracellular loop, previously analyzed by Yokoyama et al The substitution of tryptophan for arginine could con- [30], is apparently present at the cell surface. It has a ceivably alter agonist binding affinity by eliminating a reduced affinity for AVP and fails to significantly stimulate charge. Supporting this model, arginine 113 was also cAMP accumulation, even at high concentrations of ago- present in one of the AVP-photoaffinity-bound peptides nist, which is consistent with our observations. Disruption identified by Kojro et al [32]. Alternatively, the less flexible of protein folding by promiscuous inter- or intra-molecular tryptophan side chain might stearically inhibit a required disulfide bonding is one explanation for the reduced func- disulfide bond formation between the adjacent cysteine 112 tion of similar receptors with mutations that introduce new and cysteine 192 in the second extracellular loop. Work cysteines. Alternatively, data from the cognate mutation with ␤-adrenergic receptors has shown a preponderance of (F189C) in the dopamine D2 receptor suggest that this very low affinity binding sites in mutants where either residue may be implicated in the formation of an active cysteine in this bond is substituted with valine [34]. ligand binding crevice [31]. This allele retains the most function of all those tested Alleles with partial activity. Six of the alleles with missense from NDI patients. Sufficient clinical information is not variations (L81F, P95L, R113W, R181C ϩ V215M, N317K, currently available in our case to distinguish the disease and N321Y) retained some capacity to signal in response to course from that found with other mutations. Systematic 1920 Wildin et al: V2 receptor defects in NDI testing of such individuals is clearly desirable, but it has isolated L292P allele [40], suggesting that G12E is a thus far proven impractical. polymorphism. Schoneberg et al confirmed the loss of The N317K and N321Y mutations have not been previ- receptor function caused by L292P [41]. Our tentative ously reported, but also retain some signaling capacity. conclusion that C319R is responsible for the loss of recep- Surprisingly, these mutations, as well as L81F, P95L, and tor function should be confirmed by expressing it indepen- R113W, which retain function, occur in residues that are dently. completely identical across the receptor subtypes (Fig. 2). One allele in this study was obtained experimentally, not This suggests that a minimum threshold of activity is from an NDI patient. It has three different mutations in the required for evolutionary conservation at these positions, coding region, M1V, G185S, and I221T. Its near-normal and that any substitution likely reduces activity below the function seems initially surprising since the presumed ini- threshold. These residues may thus be important contact tiation codon is eliminated, and another mutation at resi- points in ligand recognition or in G protein activation. due 185, G185C, has been reported in NDI [5]. Further- An identical mutation to N317K at the cognate position more, I221T is a non-conservative, non-polar-to-polar in the hamster ␤-adrenergic receptor (N318K) has previ- change. However, a methionine is also encoded by codon 3, ously been analyzed. Agonist binding affinity was reduced presumably allowing for translation initiation there. Rela- about tenfold, whereas antagonist binding was similar to tively normal function of this receptor suggests that the first wild-type [35]. On the basis of limited maximal stimulation two amino acids of the putative coding sequence may not of adenylate cyclase, the authors later concluded that the be very important for receptor function and that codon 3 mutation also created a partial defect in G protein coupling can function as the initiator. [34]. A similar coupling defect is therefore hypothesized for Secondly, serine at position 185, in the second extracel- both N317K and N321Y in the V2 receptor. lular loop, is probably less disruptive than cysteine at the Clinical data from the diagnostic studies of subjects same position. As discussed above, the cysteine at position whose V2 receptor variants were tested (Table 2) did not 192 is believed to form a disulfide bond with C112, so a new reveal differences in urine concentrating ability that could cysteine at position 185 probably competes with C192 for be correlated with the degree of receptor activity witnessed inter-loop disulfide bond formation, disrupting secondary in vitro. structure. A new serine at this position is unlikely to have Alleles with two missense mutations. The R181C variation such an effect. was found in the same allele as a novel variation, V215 M. Threonine in place of isoleucine at position 221, the R181C has been reported three other times in NDI patients middle of the fifth transmembrane domain, involves a [3, 36, 37], once in association with a three-residue deletion change in polarity, but not in size. This third mutation may elsewhere in the same allele [3]. Experimental analysis of affect receptor function little because it is well tolerated in the two mutations separately indicated that R181C reduced the transmembrane domain. receptor function but that the three-codon deletion did not [38]. Thus, it is likely that the R181C variation is a Correlates from sequence comparisons disease-causing change in our patient as well. Distraction of Clustering of disease-associated variations is frequently the normal disulfide bonding between cysteines in extracel- ascribed to mutation “hot spots.” While a few mutations lular loops I and II has previously been suggested as a basis clearly recur, our analysis of the reported AVPR2 missense for the observed loss of function observed in the R181C mutations in NDI supports a strong bias for mutations in allele [3]. highly conserved residues of the protein. This suggests that In our patient, this change may have occurred on the among all randomly occurring mutations in this gene, those background of the V215M variation representing a rare that markedly disrupt receptor function cause clinically polymorphism. Alternatively, the V215M change could recognized disease. contribute to reduced receptor function. Interestingly, the Correspondingly, persons with mutations that change same conservative substitution (V 3 M) occurring at a other, less well-conserved residues and have a milder effect different position (residue 88) but in a similar transmem- on receptor function probably exist in the population. They brane-segment context was reported by Pan et al to repre- may only present clinically when challenged with extreme sent a polymorphism on genetic grounds [3]. Hence, water deprivation and they probably escape formal diagno- V215M may represent another rare polymorphism, but this sis. Alternatively, the may possess a secondary must be confirmed by independent functional analysis. means of water conservation that masks the effects of Two variants, G12E and C319R, were identified in functionally mild mutations in AVPR2. another patient. The G12E change has been reported previously in an allele with a different downstream mis- Conclusions sense mutation (L292P) in an NDI patient [39]. Wenkert et The documentation of a high frequency of sporadic cases al later demonstrated normal function of a synthetic allele and the demonstration of further allelic heterogeneity with only the G12E change and absent function in an provided in this study have immediate implications for the Wildin et al: V2 receptor defects in NDI 1921 diagnosis and genetic counseling of patients with congenital sin type 2 receptor gene (AVPR2) associated with nephrogenic NDI and their families. AVPR2 mutation detection and in diabetes insipidus. Nature Genet 2:99–102, 1992 6. CANFIELD MC, TAMARAPPOO BK, MOSES AM, VERKMAN AS, HOLTZ- vitro confirmation of the functional significance of muta- MAN EJ: Identification and characterization of aquaporin-2 water tions are clearly valuable techniques that should be clini- channel mutations causing nephrogenic diabetes insipidus with partial cally available, especially for the relatively common spo- vasopressin response. Hum Mol Genet 6:1865–1871, 1997 radic cases. 7. DEEN PM, VERDIJK MA, KNOERS NV, WIERINGA B, MONNENS LA, VAN OS CH, VAN OOST BA: Requirement of human renal water From a structure-function standpoint, these data support channel aquaporin-2 for vasopressin-dependent concentration of some preliminary generalizations about the V2 receptor. urine. Science 264:92–95, 1994 First, mutations occurring in receptor domains known to be 8. HOCHBERG Z, VAN LIEBURG A, EVEN L, BRENNER B, LANIR N, VAN OOST BA, KNOERS NV: Autosomal recessive nephrogenic diabetes involved in binding AVP tend to produce partial signaling insipidus caused by an aquaporin-2 mutation. J Clin Endocrinol Metab deficiencies. Second, those that alter transmembrane do- 82:686–689, 1997 mains by inserting a charged residue tend to lack receptor 9. OKSCHE A, MOLLER A, DICKSON J, ROSENDAHL W, RASCHER W, BICHET DG, ROSENTHAL W: Two novel mutations in the aquaporin-2 function regardless of the ligand concentration provided, and the vasopressin V2 receptor genes in patients with congenital presumably because they are either improperly assembled nephrogenic diabetes insipidus. Hum Genet 98:587–589, 1996 or fail to reach the cell surface. Third, mutations with only 10. VAN LIEBURG AF, VERDIJK MA, KNOERS VV, VAN ESSEN AJ, PROES- slight effects on V2 receptor function are generally not MANS W, MALLMANN R, MONNENS LA, VAN OOST BA, VAN OS CH, DEEN PM: Patients with autosomal nephrogenic diabetes insipidus found among patients with clinically significant NDI. Fur- homozygous for mutations in the water-channel gene. ther dissection of the structural and functional deficits of Am J Hum Genet 55:648–652, 1994 the mutant V2 receptors will yield valuable insights into the 11. VARGAS-POUSSOU R, FORESTIER L, DAUTZENBERG MD, NIAUDET P, DECHAUX M, ANTIGNAC C: Mutations in the vasopressin V2 receptor underlying mechanisms of G protein-coupled receptor and aquaporin-2 genes in 12 families with congenital nephrogenic function. diabetes insipidus. J Am Soc Nephrol 8:1855–1862, 1997 12. HEDIN KE, DUERSON K, CLAPHAM ED: Specificity of receptor-G ACKNOWLEDGMENTS protein interactions: Searching for the structure behind the signal. Cell Signal 5:505–518, 1993 This work was supported in part by the following grants to R.S.W.: the 13. WILDIN RS, ANTUSH MJ, BENNETT RL, SCHOOF JM, SCOTT CR: March of Dimes Birth Defects Foundation’s Basil O’Connor Award, and Heterogeneous AVPR2 gene mutations in congenital nephrogenic the National Institute of Diabetes & Digestive & Kidney ’ FIRST diabetes insipidus. Am J Hum Genet 55:266–277, 1994 Award (DK47278). Additional funding was from the Department of 14. SAMBROOK J, FRITSCH E, MANIATIS T: Molecular Cloning: A Labora- Pediatrics and the NICHD-sponsored Child Health Research Center at tory Manual (2nd ed). Cold Spring Harbor, Cold Spring Harbor the University of Texas Medical Branch, Galveston, and from the Depart- Laboratory, 1989 ment of Molecular and Medical Genetics at Oregon Health Sciences 15. BONNER TI, YOUNG AC, BRANN MR, BUCKLEY NJ: Cloning and University. expression of the human and rat m5 muscarinic acetylcholine receptor The authors wish to thank the practitioners who voluntarily referred genes. Neuron 1:403–410, 1988 patients and facilitated blood sampling: C. Ronald Scott, Israel Zelikovic, 16. MELLON P, PARKER V, GLUZMAN Y, MANIATIS T: Identification of Anne Spencer, Barbara Botelho, Laura Workman, William Smoyer, Barry DNA sequences required for transcription of the human alpha Bercu, Dr. Ruxley, Laurie Fauser, Ruth McDonald, Daniel L. Metzger, 1-globin gene in a new SV40 host-vector system. Cell 27:279–288, 1981 Kevin L. Havlik, Chris Miller, Deborah Yeh, Anthony Portale, Frank 17. MELLON PL, CLEGG CH, CORRELL LA, MCKNIGHT GS: Regulation of Boineau, Valerie Ostrower, Martin G. Bialer, Gilbert Handel, Carlos transcription by cyclic AMP-dependent protein kinase. Proc Natl Acad Bacino, Nick Morton, D.J. Carson, R. Greenberg, Yujen Edward Hsia, Sci USA 86:4887–4891, 1989 Catherine Nowak, Jay Ellison, Charles Epstein, Marc Lande, James Laub, 18. EDLUND T, WALKER MD, BARR PJ, RUTTER WJ: Cell-specific expres- Larry Greenbaum, Anita Pedersen, Yogesh Shah, Becky Finkbonner, H. sion of the rat gene: Evidence for role of two distinct 5Ј Youssoufian, Rosalie Faraci, and Ann P. Guillot. flanking elements. Science 230:912–916, 1985 19. MIGEON JC, THOMAS SL, NATHANSON NM: Differential coupling of Reprint requests to Robert S. Wildin, M.D., Mailcode L-103, Molecular and m2 and m4 muscarinic receptors to inhibition of anenylyl cyclase by Medical Genetics, Oregon Health Sciences University, 3181 SW Sam Jackson Gi-alpha and Go-alpha subunits. J Biol Chem 270:16070–16074, 1995 Park Rd., Portland, Oregon 97201-3098, USA. 20. GLANTZ SA: Primer of Biostatistics. San Francisco, McGraw-Hill, 1981, E-mail: [email protected] p 352 21. MIGEON J, NATHANSON N: Differential regulation of cAMP-mediated REFERENCES gene transcription by m1 and m4 muscarinic acetylcholine receptors. J Biol Chem 269:9767–9773, 1994 1. LOLAIT SJ, O’CARROLL AM, MCBRIDE OW, KONIG M, MOREL A, 22. BASSETT DJ: Genome cross-referencing and XREFdb: Implications BROWNSTEIN MJ: Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature for the identification and analysis of genes mutated in human disease. 357:336–339, 1992 Nature Genet 15:339–344, 1997 2. LUZIUS H, JANS DA, GRUNBAUM EG, MORITZ A, RASCHER W, 23. HOLTZMAN EJ, KOLAKOWSKI LF JR, O’BRIEN D, CRAWFORD JD, FAHRENHOLZ F: A low affinity vasopressin V2-receptor in inherited AUSIELLO DA: A Null mutation in the vasopressin V2 receptor gene nephrogenic diabetes insipidus. J Receptor Res 12:351–368, 1992 (AVPR2) associated with nephrogenic diabetes insipidus in the 3. PAN Y, METZENBERG A, DAS S, JING B, GITSCHIER J: Mutations in the Hopewell kindred. Hum Molec Genet 2:1201–1204, 1993 V2 vasopressin receptor gene are associated with X-linked nephro- 24. BODE HH, CRAWFORD JD: Nephrogenic diabetes insipidus in North genic diabetes insipidus. Nature Genet 2:103–106, 1992 America - The Hopewell hypothesis. N Engl J Med 280:750–754, 1969 4. ROSENTHAL W, SEIBOLD A, ANTARAMIAN A, LONERGAN M, ARTHUS 25. VOGEL F, MOTULSKY AG: Human Genetics: Problems and Approaches MF, HENDY GN, BIRNBAUMER M, BICHET DG: Molecular identifica- (3rd ed). Berlin, Springer, 1997, p 851 tion of the gene responsible for congenital nephrogenic diabetes 26. WILDIN R, COGDELL D: The clinical utility of direct mutation testing insipidus. Nature 359:233–235, 1992 for congenital nephrogenic diabetes insipidus. Pediatrics (in press) 5. VAN DEN OUWELAND AM, DREESEN JC, VERDIJK M, KNOERS NV, 27. NOMURA Y, ONIGATA K, NAGASHIMA T, YUTANI S, MOCHIZUKI H, MONNENS LA, ROCCHI M, VAN OOST BA: Mutations in the vasopres- NAGASHIMA K, MORIKAWA A: Detection of skewed X-inactivation in 1922 Wildin et al: V2 receptor defects in NDI

two female carriers of vasopressin type 2 receptor gene mutation. 41. SCHONEBERG T, YUN J, WENKERT D, WESS J: Functional rescue of J Clin Endocrinol Metab 82:3434–3437, 1997 mutant V2 vasopressin receptors causing nephrogenic diabetes insip- 28. VAN LIEBURG AF, VERDIJK MA, SCHOUTE F, LIGTENBERG MJ, VAN idus by a co-expressed receptor polypeptide. EMBO J 15:1283–1291, OOST BA, WALDHAUSER F, DOBNER M, MONNENS LA, KNOERS NV: 1996 Clinical phenotype of nephrogenic diabetes insipidus in females 42. OKSCHE A, SCHULEIN R, RUTZ C, LIEBENHOFF U, DICKSON J, MULLER heterozygous for a vasopressin type 2 receptor mutation. Hum Genet H, BIRNBAUMER M, ROSENTHAL W: Vasopressin V2 receptor mutants 96:70–78, 1995 that cause X-linked nephrogenic diabetes insipidus: Analysis of ex- 29. RHEE AMV, JACOBSON KA: Molecular architecture of G protein- pression, processing, and function. Molec Pharmacol 50:820–828, coupled receptors. Drug Devel Res 37:1–38, 1996 1996 30. YOKOYAMA K, YAMAUCHI A, IZUMI M, ITOH T, ANDO A, IMAI E, 43. CHEONG HI, PARK HW, HA IS, MOON HN, CHOI Y, KO KW, JUN JK: KAMADA T, UEDA N: A low-affinity vasopressin V2-receptor gene in a Six novel mutations in the vasopressin V2 receptor gene causing kindred with X-linked nephrogenic diabetes insipidus. JAmSoc nephrogenic diabetes insipidus. 75:431–437, 1997 Nephrol 7:410–414, 1996 44. FAA V, VENTRUTO ML, LOCHE S, BOZZOLA M, PODDA R, CAO A, 31. JAVITCH JA, FU D, CHEN J: Residues in the fifth membrane-spanning ROSATELLI MC: Mutations in the vasopressin V2-receptor gene in segment of the dopamine D2 receptor exposed in the binding site three families of Italian descent with nephrogenic diabetes insipidus. crevice. Biochem 34:16433–16439, 1995 Hum Molec Genet 3:1685–1686, 1994 32. KOJRO E, EICH P, GIMPL G, FARENHOLZ F: Direct identification of an 45. ROSENTHAL W, SEIBOLD A, ANTARAMIAN A, GILBERT S, BIRNBAUMER extracellular agonist binding site in the renal V2 vasopressin receptor. M, BICHET DG, ARTHUS MF, LONERGAN M: Mutations in the Biochemistry 32:13537–13544, 1993 vasopressin V2 receptor gene in families with nephrogenic diabetes 33. BIRNBAUMER M, GILBERT S, ROSENTHAL W: An extracellular congen- insipidus and functional expression of the Q-2 mutant. Cell Molec Biol ital nephrogenic diabetes insipidus mutation of the vasopressin recep- 40:429–436, 1994 tor reduces cell surface expression, affinity for ligand, and coupling to 46. OKSCHE A, DICKSON J, SCHULEIN R, SEYBERTH HW, MULLER M, the Gs/adenylyl cyclase system. Molec Endocrinol 8:886–894, 1994 RASCHER W, BIRNBAUMER M, ROSENTHAL W: Two novel mutations in 34. DIXON RAF, SIGAL IS, STRADER CD: Structure-function analysis of the vasopressin V2 receptor gene in patients with congenital nephro- the beta-adrenergic receptor. Cold Spring Harbor Symp Quant Biol genic diabetes insipidus. Biochem Biophys Res Comm 205:552–557, LIII:487–497, 1988 1994 [published erratum appears in Biochem Biophys Res Commun 35. STRADER CD, SIGAL IS, REGISTER RB, CANDELORE MR, RANDS E, 207(3):1059, 1995] DIXON RAF: Identification of residues required for ligand binding to 47. YUASA H, ITO M, OISO Y, KUROKAWA M, WATANABE T, ODA Y, the Beta-adrenergic receptor. Proc Natl Acad Sci USA 84:4384–4388, 1987 ISHIZUKA T, TANI N, ITO S, SHIBATA A, SAITO H: Novel mutations in the V2 vasopressin receptor gene in two pedigrees with congenital 36. BICHET DG, BIRNBAUMER M, LONERGAN M, ARTHUS MF, nephrogenic diabetes insipidus. J Clin Endocrinol Metabol 79:361–365, ROSENTHAL W, GOODYER P, NIVET H, BENOIT S, GIAMPIETRO P, 1994 SIMONETTI S, FISH A, WHITLEY CB, JAEGER P, GERTNER J, NEW M, ICHET RTHUS ONERGAN ENDY ARADIS DI BONA FJ, KAPLAN BS, ROBERTSON GL, HENDY GN, FUJIWARA 48. B DG, A MF, L M, H GN, P AJ, TM, MORGAN K: Nature and recurrence of AVPR2 mutations in FUJIWARA TM, MORGAN K, GREGORY MC, ROSENTHAL W, DIDWANIA X-linked nephrogenic diabetes insipidus. Am J Hum Genet 55:278– A, ANTARAMIAN A, BIRNBAUMER M: X-linked nephrogenic diabetes 286, 1994 insipidus mutations in North America and the Hopewell hypothesis. 37. KNOERS NV, VAN DEN OUWELAND AM, VERDIJK M, MONNENS LA, J Clin Invest 92:1262–1268, 1993 VAN OOST BA: Inheritance of mutations in the V2 receptor gene in 49. TSUKAGUCHI H, MATSUBARA H, ARITAKI S, KIMURA T, ABE S, INADA thirteen families with nephrogenic diabetes insipidus. Kidney Int M: Two novel mutations in the vasopressin V2 receptor gene in 46:170–176, 1994 unrelated Japanese kindreds with nephrogenic diabetes insipidus. 38. PAN Y, WILSON P, GITSCHIER J: The effect of eight V2 vasopressin Biochem Biophys Res Comm 197:1000–1010, 1993 receptor mutations on stimulation of adenylyl cyclase and binding to 50. SHOJI Y, TAKAHASHI T, SUZUKI Y, SUZUKI T, KOMATSU K, HIRONO H, vasopressin. J Biol Chem 269:31933–31937, 1994 SHOJI Y, YOKOYAMA T, KITO H, TAKADA G: Mutational analyses of 39. WENKERT D, MERENDINO JJ JR,SHENKER A, THAMBI N, ROBERTSON AVPR2 gene in three Japanese families with X- linked nephrogenic GL, MOSES AM, SPIEGEL AM: Novel mutations in the V2 vasopressin diabetes insipidus: Two recurrent mutations, R137H and deltaV278, receptor gene of patients with X-linked nephrogenic diabetes insipi- caused by the hypermutability at CpG dinucleotides. Hum Mutat dus. Hum Molec Genet 3:1429–1430, 1994 Suppl 1:S278–S283, 1998 40. WENKERT D, SCHONEBERG T, MERENDINO JJ JR,RODRIGUEZ PENA 51. TAJIMA T, NAKAE J, TAKEKOSHI Y, TAKAHASHI Y, YURI K, NA- MS, VINITSKY R, GOLDSMITH PK, WESS J, SPIEGEL AM: Functional GASHIMA T, FUJIEDA K: Three novel AVPR2 mutations in three characterization of five V2 vasopressin receptor gene mutations. Mol Japanese families with X-linked nephrogenic diabetes insipidus. Pe- Cell Endocrinol 124:43–50, 1996 diatr Res 39:522–526, 1996