The vasopressin type 2 receptor gene. Chromosomal localization and its role in nephrogenic diabetes insipidus

Regulatory Peptides, 45 (1993) 67-71

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© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00

R E G P E P 01345

The vasopressin type 2 receptor gene. Chromosomal localization and its role in nephrogenic diabetes insipidus Anita Seibold a, Walter Rosenthal a, Daniel G. Bichet b and Mariel Birnbaumer

a

aDepartment of Cell Biology, Baylor College of Medicine, Houston, TX (USA) and b Service de Ndphrologie, Centre de Recherche, Hrpital du Sacre-Coeur de Montreal and Department of Medicine, University of Montreal, Montreal (Canada)

Key words: Vasopressin receptor; Nephrogenic diabetes insipidus; Receptor mutation

Although arginine vasopressin (AVP) was first identified and named because of its pressor effects on the vascular bed, later studies found that the pathological states associated with AVP relate to its effect on water conservation in the kidney. The consequences of lack of synthesis or secretion of vasopressin were first clearly characterized when neurogenic diabetes insipidus was described (NDI), Fig. 1 [1 ]. The identification of the Brattleboro rat suffering pituitary NDI provided an accessible experimental model for this disease [2]. Molecular biology techniques applied to this problem allowed first the cloning of the genes encoding vasopressin and neurophysin II [3] and then made possible the identification of the genetic defect in the Brattleboro rat [4], and later in humans [5]. The mutations found in both species alter the amino acid composition of the precursor protein and block proteolytic processing. The recent cloning of the human V2 vasopressin receptor [6] provided the molecular tools to determine the biochemical basis of the genetic defect that causes congenital nephrogenic diabetes insipidus Correspondence to."A. Seibold, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

(CNDI), a disease characterized by lack of response of the kidney collecting duct to vasopressin in individuals that have normal responses to other hormones that act by stimulating the adenylyl cyclase pathway. The expression cloning method that we applied included the isolation of the gene encoding the vasopressin receptor, followed by the isolation of the Inherited diabetes insipidus Central (Neuro2enic) Inheritance

Autosomal dominant

Nephrogenic

Autosomal

X-linked

recessive DIDMOAD

First clinical manifestations

Variable Variable usually>1 year infancy

First 3 months of life

Mental retardation

Absent

Absent

Severe if repeated episodes of dehydration during infancy

Molecular ~terafion

VasopressinneurophysinH gen¢

Unknown

V2reeeptorgene

Fig. 1. Inherited diabetes insipidus. Symptomatology and ethiology of the disease. D I D M O A D describes the following clinical features of a syndrome: diabetes insipidus, diabetes mellitus, optic atrophy, sensorineural deafness (Peden et al., Q. J. Med. New Series, 58 (1986) 167-170).

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c D N A from a hybrid cell library and from a human kidney c D N A library. Computer simulated translation and comparison of the sequences obtained revealed the existence of two intervening sequences that interrupt the open reading frame of the human V2R gene [7]. Genetic linkage studies had localized the N D I locus to the q28-qter segment of the human X chromosome [8,9]. These data led Jans and collaborators to analyze whether mouse/human cell hybrids that contain that portion of the X chromosome may express the V2 receptor, in a manner analogous to the expression that we obtained in our transfected cell lines [ 10]. Indeed they found ligand binding activity as well as stimulation of adenylyl cyclase by AVP in the cells [ 11 ]. With this information in hand we set out to verify whether indeed the N D I gene was the V2 R receptor gene. Rather than analyzing by hybridization D N A from hybrid cells containing different chromosomes we concentrated on human/hamster cell hybrids that contain the human X chromosomes and fragments thereof [7,12,13]. We applied the polymerase chain reaction [ 14] to D N A from these cells and confirmed that indeed the V2R gene is located in the q28-qter portion of the X chromosome as illustrated in Fig. 2. The presence of the V2R gene in the Q1N cell line that lacks the DXS52 locus as shown by the absence of a hybridizing signal with the Stl4 marker [15], demonstrated that the gene encoding the receptor is adjacent but not coincidental with the DXS52 locus. The Stl4 marker has been used to define the genetic linkage of C N D I and provided the initial clues as to the location of the gene in the human X chromosome. That distinct loci are found for the Stl4 marker and the V2R gene is not surprising, since fine mapping of this region is dependent upon the recent availability of well characterized genes, replacing the use of anonymous D N A markers in mapping. The mapping in this region depicted in Fig. 2 has been further refined by the use of pulse field gel electrophoresis and probes such as human L1 CAM and V2R c D N A s (Faust, C. Sei-

Mapping

within

Xq28

.... 4.12

"i-

QIZ

+

QIN

+

Y162-Aza FRAXA

I

I DXS296

DXS295

I

II

[I111

lOS DXS304

IV DXS52

DXS15

I

V

RCP QCP G6PD

I

VI

I xqt~, F8 DXS115 DXS64

DXS305 GABRA3

Fig. 2. Diagram of mapping within the Xq28-Xqter region of the human chromosome. The diagram is an adaptation of the one recently published by Traupe et al. [15]. The distances in megabases and the division of sub-regionsin Xq28 is informationtransmitted by Dr. S.T. Warren as personal communication. The hybridization markers DXSll5, DXYS64, F8 (clotting factor 8), G6PD (glucose-6-phosphatedehydrogenase),RCP and GCP (red and green eye color pigments), DXS52, DXS15, GABRA3 (gaba A3 receptor subunit), DXS305, DXS304, and DXS296 were located according to references 7, 12 and 14. FRAXA (fragile X syndrome), DXS295 and IDS (iduronate-2-sulphatase) were located according to Refs. 13, 19 and 20.

bold, A. Bimbaumer, M. and Herman, G., unpublished data). When the genomic D N A from C N D I patients was analyzed (Southern blots) we failed to uncover any differences in the size of the V2R gene band that segregated with the disease in Bcl I, Bgl II and Tac I digests. These restriction enzymes were tested because they generate polymorphic restriction fragments in the region of the C N D I gene. The polymerase chain reaction (PCR) carried out on samples of genomic D N A obtained from these patients produced a fragment of the expected size (containing the complete open reading frame and the two introns), as well as the expected restriction pattern. The product was cloned into Bluescript and the complete open reading frame sequenced. In the first family we analyzed we found a mutation in codon 246 that arises because one of six consecutives guanosines is missing [ 16]. This deletion causes a frame shift and the premature appearance of a stop codon at 270; this predicts a receptor protein 101 amino acid shorter than the wild type receptor as illustrated in Fig. 3. It is possible that this

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V2R mutations in CNDI

1-

132 O-1. keu ~ Met Normal CTG G ~ ATG Mutant CTG E~3 ATG Leu A~p Met transversion

246

270

Q-5. Gly GlYr.~ Arg Val Normal GGG GG~ICGC... GTG Mutant GGG Gq-ICGCC . TGA Gly Gly-- Ala Stop deletion and frame shift

Fig. 3. Model and amino acid sequence of the wild type and mutant V2 receptor. Amino acid sequence of the wild-type V2 receptor presented as a model. The sites for the Q-5 and O-1 mutations are indicated. The large black circles identify the mutated codons; putative transmembrane ~t-helicesare shown at the location suggested by Kyte and Doolitle [21]; a disulfide-bridge is postulated betweenthe first and second extracellularloops based on mutational studies of Khorana et al with rhodopsin [22] and the conservation of cognate cysteines in all G protein coupled receptor. Folding of N-and C-termini and of longer intracellular loops is arbitrary. Anchoring of the carboxyl terminal portion is patterned after those of rhodopsin (two palmitoylanchors). Amino acids are given in the one-letter-code.

receptor may bind vasopressin and fail to stimulate the G protein G S, depending on whether the last two transmembrane regions contribute to the hormone binding site or are needed to define its conformation. Experiments published by Kobilka and collaborators proved that at least for the beta 2 adrenergic receptor, binding activity was not measurable when a similarly truncated m R N A was expressed in Xenopus oocytes [17]. When the V2R gene of the healthy brother of the patient was analyzed we found the expected six guanosines in this region, while the mother's genome was heterozygous: it contained one copy of the normal and one of the mutated gene. We next analyzed an unrelated patient and found

another mutation in the region encoding the open reading frame. In this family, identified as O-1, the mutation consists of a G to A transversion at codon 132 that changes the amino acid from Alanine to Aspartic acid in the putative transmembrane region three. Analyzis of other affected families applying single stranded P C R and sequencing of the regions that contain the Q-5 and O-1 mutations showed that neither mutation is present in the other pedigrees. We are sequencing the V2R genes in those families to characterize their mutations. The next step will be to characterize by in vitro expression of wild type c D N A mutated to mimic the C N D I genes the impact of those amino acid changes in the ability of the receptor to bind vasopressin and/or to couple to G s. Since in many cases mutations result in accumulation of improperly folded proteins in the endoplasmic reticulum [18], and therefore in a great reduction in the number of receptor molecules in the cell membrane antibodies will be developed to examine the effect of these mutations on the level of receptor expression concomitant with our functional studies. We hope to use the characterized C N D I mutations as a source of inspiration to start defining the portions of the V2R molecule that are responsible for vasopressin binding and stimulation of adenylyl cyclase activity. An examination of the data obtained by expressing mutated c D N A s encoding human rhodopsin may help us imagine the kind of information that can be obtained when similar experiments are done with V2 receptor c D N A mutants. The data contained in Ref. 18 show great heterogeneity in the location of mutations leading to the hereditary defect. It also shows that although it is known that 11-cis retinal binds to rhodopsin in the transmembrane region, genes containing mutations affecting one or another transmembrane segments generate proteins that bind retinal, and are able to suport the cis-trans isomerization of the ligand when excited by light. This isomerization is believed to be accompanied by a concomitant change in protein structure, thus the effect of mutations in different regions may help to pinpoint the

70 sites o f c o n t a c t w i t h the ligand. In the c a s e o f the v a s o p r e s s i n a n d o x y t o c i n r e c e p t o r s , the h y p o t h e s i s t h a t ligand b i n d i n g is d e t e r m i n e d by the first a n d

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s e c o n d e x t r a c e l l u l a r l o o p s is v e r y a t t r a c t i v e b e c a u s e s o m e a m i n o a c i d s in this r e g i o n are highly c o n s e r v e d b e t w e e n t h e m , n e v e r t h e l e s s it m a y be v e r y difficult to

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p r o v e or d i s p r o v e s u c h possibility b e c a u s e o f u n c e r tainty as to h o w m a n y s e g m e n t s are n e c e s s a r y to c r e a t e the b i n d i n g site a n d the role p l a y e d by the rest

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o f the p r o t e i n m o l e c u l e in m a i n t a i n i n g t h e s t r u c t u r e o f the l i g a n d b i n d i n g site. N e v e r t h e l e s s w i t h t h e m a terials available w e h a v e started o n o u r w a y to answer those questions.

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22 Karnik, S.S., Sakmar, T.P., Chen, H.-B. and Khorana, H.G., Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin, Proc. Natl. Acad. Sci. USA, 85 (1988) 8459-8463.