Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function

EXPERIMENTAL CELL RESEARCH 195, 478-484 (1991)

Isolation and Genetic Characterization of a Renal Epithelial Cell Mutant Defective in Vasopressin (V,) Receptor Binding and Function HEIKELUZIUS,DAVID A. JANS, PATRICIAJANS,ANDFALKFAHRENHOLZ' Max-Planck-Institut

fiir

Biophysik,

Frankfurt

am Main, Kennedy Allee 70, Federal Republic of Germany

the CAMP-dependent protein kinase [5]. The CAMPdependent protein kinase modifies the activity of a number of enzymes and the expression of specific genes through phosphorylation of a variety of protein substrates [6, 71. One approach to understanding vasopressin receptor expression and its role in signal transduction is the study of cell mutants defective in hormonal responsiveness. Several mutants of the LLC-PK, cell line have been isolated and characterized [g-11]. The Ml8 mutant [8] possesses neither vasopressin nor calcitonin binding activity, hypothesized to be the result of a single recessive mutation affecting a common step of the maturation ofboth receptors. LLC-PK, mutants affected specifically in calcitonin receptor binding, which complement the Ml8 mutant phenotype [9], have also been described. Mutations in receptor processing and metabolism have been described in other cell systems, such as the low-density lipoprotein (LDL) receptor of Chinese hamster ovary cells (CHO) where five complementation groups have been defined affecting LDL receptor function [ 12-141. A renal cell mutant defective only in the V, vasopressin receptor would be useful in studying V, receptor expression by complementation with foreign DNA encoding the receptor gene. This study describes the isolation of a new LLC-PK, mutant specifically affected in vasopressin binding activity. The VPRl mutant was selected using the photoactivatable vasopressin analogue [1-(3-mercapto) propionic acid, 8-(W-4-azidophenylamidino)lysine] vasopressin (apa-LVP) [15], which has been shown upon photoactivation under conditions leading to covalent attachment to receptor [16, 171 to elicit a prolonged stimulation of CAMP production, which is cytotoxic to LLC-PK, cells [ll, 18, 191. The VPRl mutant was found to possess negligible vasopressin binding activity, but normal calcitonin binding and responses to this and other agents elevating intracellular CAMP levels. The recessive mutation responsible for the phenotype is likely to reside in the vasopressin receptor structural gene or a gene specifically regulating V,-receptor expression.

A novel mutant of the LLC-PK, renal epithelial cell line, VPRl, was isolated after mutagenesis with Nmethyl-N-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N’-4-azidophenylamidino)lysine] vasopressin. The VPRl mutant cell line possessed less than 5% parental V, receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK, cells (wild type), VPRl cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo CAMP production, or urokinase-type plasminogen activator induction. The responses of VPRl cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5’-[@,y-imino] triphosphate, 8bromo adenosine-3’,5’-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK, and VPRl and analyzed for the dominance/recessiveness of the VPRl mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V, receptor in VPRl cells is recessive. The VPRl cell line may thus have application as a recipient for the expression of the V, receptor gene :c‘1991 Academic Press, Inc. using DNA-transfer.

INTRODUCTION The LLC-PK, porcine kidney epithelial cell line [l] is an attractive model system to investigate the function and expression of adenylate cyclase-coupled receptors. It possesses distinct receptors for the polypeptide hormones vasopressin and calcitonin [2]. Binding of these hormones results in stimulation of the membrane-associated adenylate cyclase complex [3,4] and an elevation of the intracellular CAMP level, which in turn activates

1 To whom correspondence and reprint requests should be addressed at Max-Planck-Institut fiir Biophysik, Kennedy Allee 70, D-6000 Frankfurt 70, Federal Republic of Germany. 0014.4827/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

478

RENAL

EPITHELIAL

TABLE Description

of Cells Used and Results

of Complementation

CELL

479

MUTANT

1 in Somatic

Cell Hybrids

of LLC-PK,

Cell Lines

Receptor activity Derivation

Vasopressin

Clonal parental cell line derived from LLC-PK, [l] Derived from LLC-PK, (22), carrying the plasmid pSV,neo [23] Spontaneous mutant derived from LLC-PK,, containing ouabain resistance (this study) Derived from Ml8 (81, the receptor deficient LLC-PK, mutant, carrying the plasmid pSV,neo Derived from PK4/02 as a cell line resistant to photoactivated apa-LVP (this study) Somatic cell hybrids from the parents PK4/Nl X VPRl (this study)” Somatic cell hybrids from the parents M18/N5 X VPRl (this study)’

+ +

+ +

+

+

-

+

tb

+

-c

+*

Cell line LLC-PK,/PK4 PK4/Nl PK4/02 M18/N5 VPRl (LLC-PK, x VPRl) Hybrids 1 and 5 (Ml8 x VPRl) Hybrids 1 and 16

’ For each cross a number of hybrids were derived of which two representative * Complementation of the respective mutant binding phenotype. ’ Lack of complementation.

MATERIALS

AND

METHODS

Materials. The vasopressin analogue apa-LVP was prepared as described previously [15]. Synthetic salmon calcitonin (SCT) was a gift from Dr. Riimer (SandozlBasel, Switzerland) and [“51]SCT from Prof. J. A. Fischer (Ziirich, Switzerland). Other reagents were from the sources described [8-lo]. Neomycin (G418) was from GIBCO (Eggenstein, FRG). Cell culture. The porcine kidney epithelial cell line LLC-PK, [l] and its derivative cell lines were cultured as described [8-lo]. Characteristics of the cell lines used are described in Table 1. Cell counts were performed using a modification of the method of Finlay et al. [20], with A,,, = 0.1 corresponding to lo4 cells [ll]. Isolation of a ouabain-resistant cell line. The PK4/02 cell line is a spontaneous mutant of the LLC-PK, cell line resistant to ouabain, selected using 1 M ouabain. After being selected and cloned, t,he mutant was found to stably retain ouabain resistance properties over 3 months (24 passages) in the absence of ouabain. The concentration of ouabain reducing PK4/02 viability by 90% (D,,,) was 88 f 4.0 M (n = 3), compared to 0.12 -+ 0.01 M (n = 3) for the parental LLC-PK, cell line. Isolation of VPKl cells defective in vasopressin binding activity. Logarithmically growing PK4/02 cells were exposed to 0.2 m M Nmethyl-N’-nitro-N-nitrosoguanidine for 4 h (85% cell mortality). The cells were allowed to recuperate for 4-5 days and then subjected to selection using the photoactivatable vasopressin analogue apa-LVP as described previously [ll]. Resistant cells were plated (104/100 m m plate), allowed to form colonies (30-50 cells), treated with 10 nM AVP. and then screened for nonresponding colonies using a caseinplasminogen agar overlay [8]. Vasopressin unresponsive colonies, in contrast to normal cells, fail to produce plaques of lysis due to urokinase-type plasminogen activator (uPA) production in the agar. They were picked and screened for vasopressin binding activity in a ligand plate-binding assay. The cells were grown on 24 well costar plates (diameter 25 mm) to 90-100% confluency and incubated for 30 min with [3H]AVP at 37°C. The plates were then washed six times with ice-cold NaCl/P, containing 0.5 mg/ml BSA. The cells were solubilized with NaClIP, containing 0.2% Triton X-100 and radioactivity was counted by liquid scintillation. Thirty-nine cell mutants with 2050% residual vasopressin binding activity were pooled, mutagenized,

Calcitonin

clones were examined.

and selected a second time. The screening was performed with the casein-plasminogen agar overlay and the hormone plate-binding assay (see above). One clone isolated (the VPRl line) devoid of vasopressin binding activity was cultured for further analysis. Somatic cell hybrids. Somatic cell hybrids were derived using the standard polyethylene glycol fusion technique [21]. PK4/Nl [22] is a clonal cell line derived from PK4 (a clonal cell line derived from LLCPK,) carrying the plasmid pSV,neo (231, conferring neomycin resistance. M18/N5 [9] is a clonal cell line derived from Ml8 [8] (a mutant derived after mutagenic treatment of LLC-PK,, affected in vasopressin and calcitonin receptor metabolism), carrying the plasmid pSV,neo (Table 1). Hybrids were selected in DMEM containing 1.5 PM ouabain and 1.3 mg/ml neomycin. Controls were routinely performed of individual parent cell lines subjected to the same selection conditions as the hybrids, to ensure that hybrids did not result from spontaneously occurring resistance. CAMP estimation. Determination of CAMP in cells after 5 min treatment with hormones and other agents was performed as described [18]. CAMP levels were measured using the competitive protein binding assay kit from Amersham, according to the method of Tovey et al. [24]. Measurement of adenylate cyclase. Cells to be assayed for adenylate cyclase activity were grown to 60-80% confluency in loo-mm dishes (Becton & Dickinson, Heidelberg) and, after washing with NaClIP,, were scraped into 20 m M Tris/HCl (pH 7.5) and 5 m M MgCl, [8]. Adenylate cyclase activity was measured at 30°C for 5 min in a medium containing 25 m M Bis-Tris/HCl, pH 8.3,3 m M MgCl,, 1.4 m M EDTA, 0.2 m M IBMX, 1 m M ATP, 30 U/ml creatine kinase, 20 m M phosphocreatine [25]. CAMP levels in the cell homogenates were then determined (see above). Adenylate cyclase activity is expressed as picomoles CAMP produced per minute per milligram protein. The activation constant (I&) is the concentration of hormone corresponding to 50% maximal adenylate cyclase activation above basal. Receptor binding. Specific binding of [3H]AVP and [‘251]SCT on whole cell monolayers or suspensions was determined as described previously [8]. Cell monolayers or suspensions were incubated with 10 nM [3H]AVP (lo-30 Ci/mmol) or 9 nM [‘251]SCT (330 Ci/mmol). Nonspecific binding was measured in the presence of a loo-fold excess of AVP or SCT, respectively. Hormone binding is expressed as

480

LUZIUS

femtomoles hormone bound per 10s cells. The dissociation constant (Kn) is the concentration of hormone corresponding to 50% maximal binding activity, determined from a linearizing plot according to Scatchard [25]. uPA was deAssay of urokinase-type plasminogen activator (GA). termined in a coupled assay with plasminogen and measurement of proteolytic cleavage of the S-2251 substrate (D-Val-Leu-L-Lys p-nitroanilide) (Bachem AG, Bubendorf, Switzerland) to yield a colored product (8). uPA activity is expressed in Ploug units per milligram cellular protein, using human urokinase (EC 3.4.32.31, Calbiochem, Frankfurt) as a standard. Protein determination. Protein was estimated with the dye binding assay of Bradford [26] using BSA (fatty acid free) as standard. RESULTS

of the VPRl Mutant

i

7

300

F 2 E

200 r

5 Q r

100

oL- I PK4/02

The PK4/02 cell line (Table 1) containing a dominant selectable marker for ouabain resistance was used for isolation of a vasopressin receptor deficient cell mutant. The detailed procedure is outlined under Materials and Methods. Briefly, PK4/02 cells were treated with the photoactivatable vasopressin analogue apa-LVP and irradiated with uv light [ 111. By covalent attachment of the analogue to the V, receptor [16], a persistent stimulation of CAMP production is induced in LLC-PK, cells [ 11, 181. This prolonged CAMP production is cytotoxic to LLC-PK, cells and can be used to select for resistant mutants [ll]. Resistant clones, possessing 20-50% residual vasopressin binding activity were subjected to a second round of mutagenesis followed by selection. This led to the isolation of one resistant clone (VPRl) completely lacking vasopressin binding activity. VPRl cells were cultured for further characterization. Characterization

ET AL.

Cell Line

The mutant was initially analyzed for hormone binding activity. Specific [3H]AVP binding to whole cell monolayers at different temperatures was examined (Fig. 1). VPRl cells exhibited greatly reduced vasopressin binding (0.55% of the parental value) independently of the temperature. Maximal specific [3H]AVP and [1251]SCT binding to whole cell suspensions of the VPRl and PK4/02 cell lines was then determined (Table 2). VPRl cells exhibited no vasopressin binding but essentially normal calcitonin binding as compared to the parental cell line. The mutational lesion in VPRl cells was thus specific to vasopressin receptor binding. In Vito CAMP and UPA Production Several agonists stimulate the adenylate cyclase/ CAMP system of LLC-PK, cells and induce the production and extracellular secretion of uPA [5,27]. The mutational lesion in VPRl was further examined by treating cells with hormone or agents elevating intracellular CAMP concentration. These incubations were carried out in the presence or absence of the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) which amplifies the intracellular CAMP level by blocking CAMP

VP41

PK4/02

VPRI

4Y

23oc

( 120 m,n i

j 120 min ’

PK4/02

VPRI 37OC

I30

min i

FIG. 1. Specific binding of 13H]AVP by PK4/02 and VPRl cells. Cell monolayers (80-90% confluent) were washed and incubated with 10-s M [3H]AVP in the absence and presence (nonspecific binding) of a loo-fold excess of AVP for 120 min at 4 or 23°C and for 30 min at 37°C (maximal binding). Separation of membrane bound from internalized radioactivity was achieved by acid washing. Unbound ligand was removed by washing at 4°C subsequent to binding, and cells were lysed in 0.1% Triton X-100 in NaClIP,. Cell extracts were both counted for radioactivity and analyzed for protein concentration. The cell-associated radioactivity is expressed as specific (0) and internalized (0) binding. Results represent the means of a single typical experiment performed in duplicate.

degradation. VPRl cells treated with AVP with or without IBMX showed no CAMP-production (Figs. 2A and 2B). The responses of VPRl to SCT or to the adenylate cyclase activator forskolin (FSK) were comparable to or higher than that of the parental PK4/02 cell line (Figs. 2A and 2B), indicating functional adenylate cyclase and calcitonin receptors in the cell line. VPRl cells produced 4280 and 5105 pmol cAMP/mg after 5 min treat-

TABLE

2

Specific Binding Activity of [3H]AVP of the Cell Lines PK4/Nl (Normal), MlWN5 (Mutant), VPRl (Mutant), and Somatic Cell Hybrid Derivatives Specific binding [fmol/106 cells] Cell line PK4/Nl MlWN5 VRPl (LLC-PK, X VPRl) Hybrid (LLC-PK, X VPRl) Hybrid (Ml8 X VPRl) Hybrid 1 (Ml8 X VRPl) Hybrid 16

[3H]AVP

1 5

157 4 7 17 124 4 7

[‘251]SCT 168 2 189 180 145 130 173

Note. Maximal specific binding was determined on EDTA-suspended cells as described under Materials and Methods. Data represent the means of a single typical experiment performed in triplicate.

RENAL

EPITHELIAL

CELL

481

MUTANT

7

E G

100

5000

E

Cl0

LOO0

a z

60

3000

2

40

2000

3 g

20

1000

; -

0

,

0 PK4/02

VPRl

PK4/02

VPRl

FIG. 2. Production of intracellular CAMP by the parental PK4/ 02 and the mutant VPRl cell lines. Monolayers of cells (6OW30% confluent) were washed twice with NaCl/Pi, and then incubated for 5 min in the absence (A) or presence (B) of 0.5 n&f IBMX without addition (k.) or with 90 nM AVP (m), 30 r&f SCT (Cl), or 0.1 mM FSK (Q. Results represent the means * SD for at least two separate determinations performed in duplicate.

ment with SCT or forskolin in the presence of IBMX whereas PK4/02 cells produced 2165 and 1735 pmol/ mg, respectively. These results were further examined by investigating uPA production in response to agents elevating the intracellular concentration of CAMP. Medium was sampled after 8 h (Fig. 3). The mutant produced negligible amounts of uPA in response to AVP. The responses of VPRl cells to SCT, forskolin, and the CAMP analogue 8-bromo adenosine-3’,5’-monophosphate (BrcA, a direct activator of the CAMP-dependent protein kinase) were comparable to those of PK4/02. The production of uPA can also be induced in LLC-PK, by phorbol myristate acetate (PMA, an activator of calcium-phospholipid-dependent protein kinase) [ 10, 281. The uPA response of VPRl cells was also comparable to that of the parental cell line (Fig. 3), indicating the specificity of the lesion affecting the vasopressin response in VPRl to the CAMP system.

0-L

I

-10

I

-G

-8 log

[AVP]

iM1

FIG. 4. Concentration dependence of adenylate cyclase activation by AVP in cell homogenates ofthe PK4/02 (0) and VPRl(0) cell lines. Cell homogenates were incubated with AVP of increasing concentrations, and adenylate cyclase activity was measured as described previously [S, 241. Results represent the mean -+ SD for a single typical experiment performed in triplicate.

In Vitro Adenylate

Cyclase Activities

The activation of adenylate cyclase in cell homogenates of PK4/02 and VPRl cells was examined subsequent to treatment with hormones and other CAMPstimulating reagents. Figure 4 shows the adenylate cyclase activity of PK4/ 02 and VPRl cells in response to various concentrations of AVP whereby VPRl was completely insensitive to stimulation. The concentration of AVP stimulating half-maximal activation of adenylate cyclase in PK4/02 was 1 nM, comparable to the receptor affinity for ligand (Fig. 7). The basal adenylate activity and the activities found in the presence of SCT, forskolin, NaF (an activator of the G, subunit), or the nonhydrolyzable analogues Gpp(NH)p and GTP,S in VPRl cell extracts were com-

_--.600

PK L/02

VPRI

FIG. 3. uPA production by the parental PK4/02 and the mutant VPRl cell lines. Cells were incubated without addition ((IIII) or with 100 r&f PMA (m), 10 n&f AVP (m), 10 nM SCT (O), 0.1 mM forskolin (W), or 1 n&f BrcA (E21).After 8 h media were collected and UPA activity was measured as described under Materials and Methods. Data are the mean f SD of a single typical experiment performed in triplicate.

PK 4/!12

vPR1

FIG. 5. Adenylate cyclase activity in PK4/02 and VPRl cell homogenates. Cell homogenates were incubated for 5 min at 30°C without addition (UiJ) or with 0.1 pM AVP @7/J),0.1 pM SCT (O), 10 pM forskolin (a), 10 m&f NaF Cm), 0.1 m&f Gpp(NH)p (fJY), or 0.1 m&f GTPyS (8). Adenylate cyclase activity was measured as described 18, 241. Results are the mean + SD for a single typical experiment performed in triplicate.

482

LUZIUS

ET AL.

FIG. 6. Agar screening test for uPA production by somatic cell hybrids (PK4/Nl X VPRl) (A), VPRl (B), and PK4/Nl (C) stimulated with AVP. Cells were plated (5 X lo5160 m m plate) in medium containing ouabain and neomycin, allowed to form colonies (SO-50 cells), treated for 3 h with 10 nM AVP, and then screened for responding colonies using a casein-plasminogen agar overlay.

parable to parental (Fig. 5). It was concluded that both the catalytic subunit and the regulatory (Gs) component of adenylate cyclase were normal in the VPRl cell line.

Somatic Cell Hybrids To investigate genetically the VPRl lesion affecting vasopressin receptor function, somatic cell hybrids were derived by fusion of the cell lines VPRl (mutant) and PK4/Nl (parental) and of the mutant cell lines VPRl and Ml&J. Ml8 is a LLC-PK, mutant lacking both vasopressin and calcitonin binding activity [S, 91. Table 1 summarizes the derivation of the somatic hybrid cell lines, as well as the results for complementation of receptor function. The somatic cell hybrids were selected in medium containing ouabain and neomycin. Surviving colonies were screened for uPA production in response to AVP using the casein-plasminogen agar overlay technique [8] (Fig. 6). In contrast to VPRl cells, the somatic cell hybrids formed between PK4lNl and VPRl recovered

uPA production in response to AVP (Fig. 6A). These cells were cultured for further characterization. The analysis for the dominance/recessiveness of the VPRl phenotype was performed by measuring hormone binding and in uiuo CAMP production of the somatic cell hybrids. As shown in Table 2 and Fig. 7, the hybrid cell lines 1 and 5 (LLC-PK, X VPRl) exhibited approximately parental binding activity in terms of both maximal binding capacity and KD. The binding affinity of [3H]AVP for the vasopressin receptor of the cell line PK4/Nl was 1.1 nM and for the hybrid cell line 1 (LLCPK, X VPRl) 1.3 nM. It was accordingly concluded that the mutation responsible for lack of AVP binding in the VPRl cell line is recessive. The fusion between VPRl and Ml8 was performed to test whether the mutations affecting the vasopressin receptor in VPRl and Ml8 resided in the same gene. Calcitonin binding in the hybrid cell clones 1 and 16 (Ml8 X VPRl) was comparable to that of the parental cell line LLC-PK,. In contrast, vasopressin binding was not complemented (Table 2). To confirm functional complementation of the V, re-

RENAL

EPITHELIAL

CELL

483

MUTANT

DISCUSSION

0.0 0

-6 log

concentratton

free

F3H] AVP

(MI

FIG. 7. Specific binding of [3H]AVP to whole cells of PK4/Nl (0) or the somatic cell hybrid 1 (LLC-PKixVPRl) (0). Cells (2 X 10e6/ml) were incubated with increasing concentrations of [3H]AVP in the absence or presence (nonspecific binding) of a 100. fold excess of AVP for 30 min at 30°C. Data are the mean k SD for a single typical experiment performed in triplicate.

ceptor defects in the hybrid cell lines, CAMP production in response to hormonal treatment was assessed. As expected, the hybrid cell line 1 (LLC-PK, X VPRl) showed CAMP production response to AVP, SCT, or forskolin (Fig. 8). The responses were increased compared to LLC-PK, cells (Fig. 8), possibly as the result of abnormal expression of various membrane components of the CAMP system in the hybrid cell line. Treatment of the hybrid cell line 16 from the mutant X mutant cross (Ml8 X VPRl) with AVP resulted in no detectable CAMP production. In contrast, the calcitonin receptor defect of Ml8 was complemented, whereby CAMP production was induced in response to XT. The responses to SCT and in particular forskolin were increased relative to all other cell lines (Fig. 8). The reason for this is unclear, but may again relate to abnormal gene expression in the hybrid cell line, whereby, in the absence of functional vasopressin receptors, an excess of G-protein components is able to interact with calcitonin receptors or the cyclase catalytic subunit itself. Functional complementation of calcitonin but not vasopressin responsiveness was observed between the VPRl and Ml8 mutants, implying that both mutants are affected in the same gene affecting the vasopressin receptor.

This study describes the isolation and characterization of a new cell mutant VPRl specifically devoid of vasopressin binding activity. Selection was performed using a photoactivatable vasopressin analogue which has been shown to elicit a persistent stimulation of CAMP production by adenylate cyclase in LLC-PK, cells, presumably by bypassing the normal cellular down-regulation processes through receptor internalization and dissociation of the ligand from receptor in lysosomes [ll, 191. The isolated cell line, VPRl, possesses negligible vasopressin binding activity, and lack of response to hormone in terms of in vivo CAMP production, adenylate cyclase activation, and uPA induction. VPRl is able to respond to all nonvasopressin-receptor-mediatedstimulation of in vivo CAMP production, in vitro adenylate cyclase activation, and uPA production. Responses to PMA were also normal. In fact, VPRl cells showed increased intracellular CAMP production in response to SCT or forskolin, which may be attributable to a secondary effect of the vasopressin receptor defect which may influence the expression and function of other components of the CAMP system. VPRl cells contain less than 5% residual vasopressin binding activity compared to parental cells. This could result from [3H]AVP binding by the oxytocin receptors of LLC-PK, cells (3.2 fmol/ lo6 cells) [29]. The impairment of vasopressin binding appears to be due to a reduction in the number of receptor binding sites, but also may involve a drastic reduction in the receptor affinity for the hormone. The mutation in vasopressin receptor function in VPRl is recessive, since somatic cell hybrids formed between the parental cell line and VPRl retain normal levels of binding activity and CAMP production in response to vasopressin. In contrast, the fusion between

2

PK4INl

PK4/Nl I VPRI Hybrid 1

VPRl

M18/NS I VPRl Hybrid 16

MlB/NS

FIG. 8. Production of intracellular CAMP by PK4/Nl, VPRl, MlWN5 cells, and somatic cell hybrid derivatives. Monolayers of cells (60-80% confluency) were washed twice with NaClIP,, and then incubated for 5 min in the presence of 0.5 m&f IBMX without adcltion (lJ@,with 90 nM AVP @I), 30 nM SCT (O), or 0.1 mM FSK (a). Results represent the means + SD for at least two separate determinations performed in duplicate.

484

LUZIUS

two mutants, VPRl and Ml8 (lacking vasopressin and calcitonin receptor binding), only showed complementation of calcitonin binding. It was concluded that Ml8 contains two independent recessive mutations, of which the vasopressin binding defect occurs in the same gene as that in VPRl. This would indicate that previous conclusions concerning the Ml8 cell line as containing a single mutation in a biosynthetic step in the common pathway of calcitonin and vasopressin receptors [8] is incorrect. That recessive mutants affected in V,-receptor function are apparently isolated with relatively high frequency in the LLC-PK, diploid cell line [this study and 8, 91 is consistent with the recent data that the vasopressin V,-receptor is probably encoded by the X-chromosome [30]. The LLC-PK, cell line is derived from a male pig [l] and hence haploid for the V,-receptor gene. Since the mutation affecting vasopressin binding activity in VPRl is recessive, it could be used to clone the vasopressin receptor structural gene via the complementation of the mutant phenotype through introduction of foreign DNA using transfection. Screening of a large number of transfected VPRl cells could be examined for complementation using a casein-plasminogen agar overlay assay (see Materials and Methods). Sardet et al. [31] have employed a similar approach to clone the Na+/H+-antiporter by complementation of a defective cell mutant. Once the vasopressin receptor gene has been cloned, it will be of interest to determine the amino acid substitution constituting the mutation of VPRl. The examination of this and similar mutants in vasopressin receptor function at the molecular level will assist in delineating the structural domains of the receptor important for ligand binding, biosynthesis, and Gprotein mediated signal transduction. The authors thank Drs. Elzbieta Kojro and Josef Zsigo for synthesis of apa-LVP, Dr. Brian A. Hemmings and CIBA Geigy (Basel) for making the Ml8 and PK4/Nl cell lines available, Dr. Rijmer (Sandoz A. G., Basel, Switzerland) for the gift of SCT, Professor Jan A. Fischer (University of Zurich, Switzerland) for the gift of [‘251]SCT, and Mirek Jurzak for helpful discussions. This work, which is part of the Ph.D. thesis of H.L. (Universitat Frankfurt), was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 169) to F.F.

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22. 23. 24. 25. 26. 27. 28. 29.

30.

12,670-677. 2.

Dayer, J.-M., Vassalli, J.-D., Bobbit, J. L., Hull, R. N., Reich, E., and Krane, S. M. (1981) J. Cell Bid. 91, 195-200.

Received March 6, 1991

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