BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]460-1468

Vol. 180, No. 3, 1991 November 14, 1991

THE ORPHAN RECEPTOR cDNA RDC4 ENCODES A 5-HTID SEROTONIN R E C E P T O R C. M a e n h a u t 1, J. Van Sande 1, C. Massart 1, C. Dinsart 1, F. Libert 1, E. M o n f e r i n i 3, E. Giraldo 3, H. Ladinsky 3, G. Vassart 1-2, and J.E. Dumont I 1 Institut de Recherche Interdisciplinaire, Facult~ de M~decine,

2 Service de G~n~tique,

Universit~ Libre de Bruxelles,

route de Lennik, 3 Dept of Biochemistry,

1070 Bruxelles,

Boehringer

Campus Erasme,

808

Belgium

Ingelheim,

Italia S.P.A.,

Via Serio 15, 20139 Milano, Italy Received September 25, 1991

The c D N A of RDC4, a putative receptor of the G protein-coupled r e c e p t o r family, has been cloned by PCR methodology. The primary structure of this receptor showed h o m o l o g y with the serotonin 5-HTIA receptor. In this work, RDC4 m R N A has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 c D N A has been transfected t r a n s i e n t l y in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the t r a n s f e c t e d cos-7 cells using [3H]-LSD showed a pattern of drug a f f i n i t i e s c o n s i s t e n t with the known p r o p e r t i e s of a 5-HTID receptor. RDC4 t h e r e f o r e codes for a 5-HTID r e c e p t o r which in the s£udied systems is p o s i t i v e l y coupled t o - a d e n y l a t e cyclase. ©1991AcademicP..... Inc.

The G p r o t e i n - c o u p l e d

receptors

constitute

a family of proteins

e n c o d e d by genes w i t h a common a n c e s t o r

(i, 2).

monomeric

transmembrane

structure

The e v o l u t i o n a r y exploited sequence series ligands

with seven p u t a t i v e

relationship

of

of G p r o t e i n - c o u p l e d

to clone new members similarities "orphan"

of this

(3, 4, 5).

receptors

They share a receptors

has been

gene family by relying on their

The result

awaiting

domains.

is the accumulation

the identification

of a

of their

(4, 6, 7, 8).

A cloning

s t r a t e g y using the p o l y m e r a s e

of d e g e n e r a t e

primers

chain reaction with a couple

allowed us to clone an orphan receptor

from a

A b b r e v i a t i o n s : 5-HT : 5-hydroxytryptamine; 8-OH-DPAT : 8-hydroxy-2(di-n-propyl- amino) tetralin; 5-MeOT : 5-methoxytryptamine; LSD : lysergic acid diethylamide; TFMPP : m - t r i f l u o r o m e t h y l p h e n y l p i p e r a z i n e ; IBMX : isobutylmethylxanthine. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

1460

Vol. 180, No. 3, 1991

dog t h y r o i d

c D N A library,

HTIA receptor RDC4 encodes subtype

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(4).

RDC4,

presenting

In the present

a receptor

which activates

study we show that the orphan c D N A

having the b i n d i n g adenylate

a strong homology to the 5-

properties

of a 5-HTID

cyclase.

M A T E R I A L S AND M E T H O D S cDNA and RNA c o n s t r u c t s : To obtain recombinant RDC4 mRNA, the c o d i n g sequence of RDC4 was amplified by PCR using primers d e s i g n e d to introduce a Bgl II r e s t r i c t i o n site on each side of the RDC4 coding sequence, and the c D N A was cloned in the Bgl II site of pSP64T (9). B o e h r i n g e r SP6 p o l y m e r a s e kit, adapted to p e r f o r m simultaneous capping, was used to produce RDC4 m R N A in in vitro transcription. The m R N A was r e s u s p e n d e d in water for the injections. For the t r a n s f e c t i o n experiments, the coding region of RDC4 c D N A was a m p l i f i e d by PCR and inserted into the ECO RI Bam HI sites of pBSK +. The Xho I Bam HI fragment of this pBSK + RDC4 p l a s m i d was then subcloned in the e x p r e s s i o n v e c t o r pSVL. The resulting construct pSVL/RDC4 was used for transient t r a n s f e c t i o n in cos-7 cells. E x p r e s s i o n systems : Y1 cells (Flow Laboratories, Irvine, UK) were grown and m i c r o i n j e c t e d as described (10). During the experiments, the cells were kept in K r e b s - R i n g e r bicarbonate m e d i u m s u p p l e m e n t e d with 8 mM glucose and 0.5 g/l albumin. The agents were added 30 min after injection of RDC4 m R N A (0.2 mg/ml), and m o r p h o l o g i c a l rounding up of the cells was o b s e r v e d after 2 hours. For transient expression, cos-7 cells were t r a n s f e c t e d with pSVL/RDC4 DNA using the D E A E - d e x t r a n method. Cells were h a r v e s t e d 72 hours after transfection. cAMP d e t e r m i n a t i o n s : cAMP was d e t e r m i n e d using a r a d i o i m m u n o a s s a y (RIA) method, as d e s c r i b e d (11). M e m b r a n e p r e p a r a t i o n and b i n d i n g assay : T r a n s i e n t l y t r a n s f e c t e d cos-7 cells were h a r v e s t e d in cold p h o s p h a t e - b u f f e r e d saline (PBS), lacking Ca ++ and Mg ++. The cells were r e s u s p e n d e d in 15 mM Tris-HCl (pH 7.4) c o n t a i n i n g 2 mM MgCI2, 0.3 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 ~M leupeptine, h o m o g e n i z e d in a glass Dounce h o m o g e n i z e r and c e n t r i f u g e d at 40,000 x g for 30 minutes. The final pellet was r e s u s p e n d e d in 50 mM Tris HCI (pH 7.4) c o n t a i n i n g 10 ~M pargyline and 0.1% ascorbic acid. All the e x p e r i m e n t s were p e r f o r m e d in triplicate in a final volume of 200 ~i (10 ~i of [3H]-LSD, 20 ~i of c o m p e t i n g drug, 70 ~i of assay buffer and 100 ~i of membrane suspension at a p r o t e i n c o n c e n t r a t i o n of 0.75 or 1 ~g/~l). Incubation was stopped by the addition of 5 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) and rapid v a c u u m f i l t r a t i o n t h r o u g h GF/B glass fiber filters (Whatman) and two subsequent 3 ml washes. Filters were then introduced into s c i n t i l l a t i o n vials, w i t h 4 ml of Filter Count (Packard). Non specific binding was d e t e r m i n e d with 10 ~M 5-HT and amounted to about 10-20% of the total binding. [3H]-LSD a s s o c i a t i o n was e v a l u a t e d at 25°C at different times (0 -120 min) with 6.5 nM [3H]-LSD. D i s s o c i a t i o n of [3H]-LSD was started after incubating 6.5 nM [3H]-LSD with membranes at 25°C for 30 min. At this time (time 0), 10 ~M 5-HT was added and determinations were made at various intervals over a 60 min period. Saturation studies were p e r f o r m e d by i n c u b a t i n g at 25°C for 30 min the membranes in the presence of v a r i o u s concentrations (0.625 - 90 nM) of [3H]-LSD. For c o m p e t i t i o n studies, membranes were incubated at 25°C for 30 min in the presence of 5 - 7 nM [3H]-LSD and different c o n c e n t r a t i o n s of u n l a b e l e d ligands. S a t u r a t i o n and competition experiments were analyzed using the LIGAND p r o g r a m (12, 13) to obtain a f f i n i t y and Bmax values. Hill c o e f f i c i e n t s (nH) were c a l c u l a t e d by linear r e g r e s s i o n 1461

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

analysis and a s s e s s e d for significant deviation from unity (Student t test). All e x p e r i m e n t s were carried out at least 3 times on different preparations. C h e m i c a l s : [3H]-LSD (62.5 - 65 Ci/mmol) and [3H]-5-HT (28 Ci/mmol) were p u r c h a s e d from N e w England Nuclear (Boston, MA), 8-0H-DPAT from Research B i o c h e m i c a l Incorporation (Wayland, MA), TFMPP from A l d r i c h Chimica Srl (Milan, Italy), 5-HT, 5-MeOT, m i a n s e r i n and (-)propranolol from Sigma Chemical Co. (St Louis, MO). K e t a n s e r i n and s u m a t r i p t a n were gifts respectively from J a n s s e n P h a r m a c e u t i c a (Beerse, Belgium) and Glaxo Group Research (Greenford, UK). M e s u l e r g i n e and m e t h y s e r g i d e were gifts from Sandoz Ltd. (Basel, Switzerland). RU 24969, buspirone and ICS 205-930 were synthesized at B o e h r i n g e r I n g e l h e i m Italia (Milan, Italy). All other reagents were from c o m m e r c i a l sources. RESULTS Microinjection

enhance

cAMP a c c u m u l a t i o n

to retract As RDC4

appeared

serotonin.

i).

10 -7 M,

in Y1 adrenocortical

from the monolayer

microinjected

M,

of RDC4 m R N A in Y1 a d r e n o c o r t i c a l

and to assume

cells

Incubation

a rounded m o r p h o l o g y

10 -8 M) resulted

The m o r p h o l o g i c a l

(10).

the cells

for sensitivity

of the m i c r o i n j e c t e d

that

cells cause the cells

c l e a r l y related to the 5-HT receptor,

w i t h RDC4 m R N A were assayed

: Agents

to

cells with serotonin

in a rapid r o u n d i n g

changes were identical

up of the cells

to those observed

(10 -6 (Fig. in the

Figure 1. Microinjection of RDC4 receptor mRNA in Y1 cells. Waterinjected (left~ and mRNA-injected (right) cells were incubated with serotonin (10- M) in the presence of 10 -6 M Ro-201724 and 10 -6 M isobutylmethylxanthine.

1462

V o l . 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4O no addition + 5 - H T I~M 55

~

~

+ 8-0H-DPA7

I~M

30-

5

25

ck 2O

15

Non-tronsfected

RDC4-transfected

cos cells

cos cells

Figure 2. cAMP measurements

in non transfected and in RDC4 transfected

cos-7 cells. 72 h o u r s a f t e r transfection, the cells f o r 30 m i n w i t h 5 - H T ( 1 0 - 6 M) o r 8-OH-DPAT ( 1 0 - 6 M ) , o f 10 - 3 M R o - 2 0 1 7 2 4 a n d 10 - 3 M i s o b u t y l m e t h y l x a n t h i n e .

indicate a representative triplicates

experiment out of 4 (average _+ SEM of

).

presence

of a g e n t s

response

to d o p a m i n e

concentrations

elevating

cAMP

levels

(10 -4 M).

The agents

were

with phosphodiesterases

and

had no effect

which

appeared

after

30 m i n

the same

dish,

which

other

RNA,

reached

remained

were

level

results

suggested

that RDC4

coupled

to a d e n y l a t e

the

stimulation

transiently the cells

about

transfected

cellular

w i t h pSVL/RDC4.

norepinephrine

(10 -6 M),

or 8-OH-DPAT

treatment

cos-7

cells

increase. failed

was not able transfected to the

levels.

5-HTIA

type

with water

in

or w i t h

The r e t r a c t i o n These

positively

levels

Taking

Treatment

cells,

synthesis cos-7

with

the 5 - H T - i n d u c e d suggesting

that

of 5-HT receptors. 1463

cAMP

that o n l y

represents cos-7

cAMP

A small

increase

but

2 led to

1-10%

of

a

cells w i t h

levels.

RDC4 p r o b a b l y

of

Fig.

10 -6 M 5-HT

account

of u n t r a n s f e c t e d

intracellular

transfection,

(10 -7 , 10 -6 M),

in the p r e s e n c e

this

to

cells w e r e

w e r e measured.

cells into

in r e s p o n s e

after

5-HT

(10 -6 M),

to be transfected,

to increase

to r e p r o d u c e cos-7

and cAMP

with

of the t r a n s f e c t e d

in cAMP

are e x p e c t e d

substantial 10 pM 5-HT

IBMX,

cAMP

72 hours

for 30 m i n u t e s

that

changes

Y1 cells

conditions.

c o d e d by RDC4,

incubated

shows

the

after microinjection.

were

a 39% i n c r e a s e

while

could be a 5-HT r e c e p t o r

: To evaluate

and

after

(10 -6 M R o - 2 0 1 7 2 4

cyclase.

of the p r o t e i n

10 -3 M R o - 2 0 1 7 2 4

cells,

or i n j e c t e d

these

2 hours

30 min.

A partial

o n l y at h i g h

The m o r p h o l o g i c a l

not i n j e c t e d under

observed

inhibitors

per se.

unaffected

was

added

in the m i c r o i n j e c t e d

a maximum

cAMP m e a s u r e m e n t s

such as forskolin.

and to n o r e p i n e p h r i n e

microinjections, IBMX),

were incubated in the presence The data shown

1 or

8-0H-DPAT in RDC4

does

not b e l o n g

significant

increase

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o

100

o rO

EL

(D

E

T 0.3]...

.0

o

I

r=o.9

"0.fl

z

"%

~

50.

,~O 0.5-

b0

v 3_

~o

0,0

2b

0

®

0.0

0.5 1.0 Specific binding (pmol mg- l pr otei n)

4'0 60 [3H]LSD (nM)

1.5 ~

80

160

~ (k~,)

-I0

-9

-§

~"

-7 -6 -5 log drug (M)

-~1

-3

Figure 3. Specific binding of [3H]-LSD (0.6-90 nM) to transiently transfected cos-7 cells membranes. Inset: scatchard plot of the data. The data show one representative experiment out of 4, performed with triplicates. Figure 4. Competition binding curves for [3H]-LSD binding (5-7 nM) of various drugs (sumatriptan (D), 5-MeOT (m), 5-HT (~), RU 24969 (A), 8OH-DPAT (o), mesulergine (e). The results represent typical experiments performed at least three times in triplicate. The affinity values for the drugs are given in table I.

in cAMP was also o b s e r v e d mRNA

(not shown)

in Xenopus

oocytes

and treated with 5-HT.

microinjected

No significant

with RDC4

increase

in

inositol p h o s p h a t e s was measured after 10 and 30 min 5-HT stimulation in t r a n s f e c t e d cos-7 cells, in experiments in which a positive cyclic AMP response

was obtained.

Binding experiments transfected (8-10 nM) cells.

cos-7

and

available

[3H]-LSD

cells.

tracer

radioligand. after min

specific

no b i n d i n g

low compared

At 25°C,

at 25°C.

[3H]-LSD.

the specific

the association

The c a l c u l a t e d

± 0.49 p m o l e / m g

Scatchard

analysis

(Fig.

class of sites with Hill Figure

(n=4)

3, inset) slopes

4 shows c o m p e t i t i o n

were performed

site

(6.3 nM)

for 30

is similar to

(14).

Bmax

and a v e r a g e d

for these membrane preparations. the presence

close to unity

1464

of a single

(0.90 ± 0.05, n=4).

curves of various

3

binding of

of transfection

revealed

binding

specific

± 3.9 nM, n=4)

upon the e f f i c i e n c y protein

[3H]-LSD.

[3H]-LSD as the

showed an half time of about

(ii.i

found for the 5-HTID

were dependent

with

obtained

[3H]-LSD reached e q u i l i b r i u m

the saturable

K D value

in

activity of the

performed of

[3H]-5-HT

from transfected

to those obtained with

were therefore

3 displays

displaced

binding values

F u r t h e r binding experiments Fig.

the affinity value values

binding

on RDC4

sites were detected

Due to the lower specific

studies

(not shown).

were performed

10 ~M serotonin

30 to 40 min and the d i s s o c i a t i o n

minutes

1.44

(5-7 nM)

(28 Ci/mmole),

[3H]-5-HT were

Further b i n d i n g

experiments

cells membranes.

For both ligands,

untransfected

with

: Binding

drugs against

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Affinity values

Table I of various drugs for RDC4, recognition sites

DRUG

RDC4 Ki (nM)±

5-MeOT Sumatriptan LSD 5-HT RU 24969 8-0H-DPAT Mesulergine (-) propranolol ICS 205-930 Ketanserin

SEM

RDC4 pK i

7.0 -+ 2.15 8.9 -+ 1.39 ii.i + 3.9 17.5 ± 4.82 65.6 ± 11.6 975 -+ 42 13010 ± 2361 15092 -+ 1964 > 100000 = 1000

8.2 8.1 7.9 7.8 7.2 6.0 4.9 4.8 < 4.0 = 6.0

5-HTIB , and 5-HTID 5-HTIB pK i

5-HTID pK i

6.4 (a) 6.4 6.8 7.6 8.4 4.2 4.9 7.3 5.7

8.2 (b) 7.5 8.2 8.4 7.3 5.9 5.2 5.5 < 5.0 (c) 6.0

Values represent the mean ± SEM of at least three separate experiments performed in triplicate. Hill coefficients for the competing drugs did not differ from unity. Data for 5-HTIB and 5-HTID sites are from Hoyer and Schoeffer (14) (a) from Hoyer et al. (20~ (b) from Hoyer et al. (21) (c) from Herrick-Davis and Titeler (22). [3H]-LSD,

and t a b l e

dopamine

nor n o r e p i n e p h r i n e

to d i s p l a c e 8-0H-DPAT, 205-930,

the

compounds

it w a s

of s o l u b i l i t y

[3H]-LSD

receptor

was a 5 - H T I B o r 5 - H T I D drugs

tested

rather

than a 5-HTIB

(Fig.

5) was o b s e r v e d

5-HT2,

binding, subtype

(table

between

compatible

affinities

in t r a n s f e c t e d

cos-7

cells membranes

in t i s s u e m e m b r a n e s .

No significant

RDC4

and 5-HTIB

receptor

Ki

As

to f u l l y t h a t the R D C 4

investigated

receptor

the

(I ~M) and

experiments

receptor

found between

a correct

(15,

16).

with a 5-HTID

I). A h i g h c o r r e l a t i o n

the binding

that

5 - H T 3 or 5 - H T 4 s u b t y p e

R U 24969

was

able of

ICS

to c a l c u l a t e

the p o s s i b i l i t y

gave pK i values

subtype

ketanserin,

at h i g h c o n c e n t r a t i o n s .

(not shown),

specific

10 -4 M) w e r e

The l o w a f f i n i t i e s

impossible

able in p r e l i m i n a r y

the

Neither

r u l e d out the p o s s i b i l i t y

problems

(10 ~M)

(I ~M) w e r e

tested.

10 -5 M,

mesulergine,

(not shown)

displace

The various

10 -6 M,

(not shown).

is of the 5 - H T I A , 5-HTIc,

like T F M P P

sumatriptan

(10 -7 M,

binding

For ketanserin,

because

the K i of the d r u g s

(not shown),

metoclopramide

15).

value

[3H]-LSD

buspirone

RDC4 r e c e p t o r (14,

I displays

(r=0.96)

of the RDC4 c o d e d

and those

for the 5 - H T I D

correlation

(r=0.38)

was

affinities.

DISCUSSION

We report receptor mRNA

here the pharmacology encoded

in Y1 c e l l s

cells.

and f u n c t i o n a l

b y t h e c D N A RDC4 e x p r e s s e d and by transfection

The pharmacological

coupling

of the o r p h a n

by microinjection

profile

of RDC4 c D N A c o n s t r u c t s

1465

of the e x p r e s s e d

receptor

of R D C 4 in c o s - 7 was

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8 ¸

8

7

7¸

v {D. nq

~6

/

I

LO

uo

r=0.38

5

4 4

9

4

5

6

RDC4 (pKi)

7

8

RDC4 (pKi)

Figure 5. Correlation between RDC4 and 5-HTIB , 5-HT D recognition sites. Data are taken from table I, excluding ICS 205-930 and ketanserin. defined from b i n d i n g studies performed on RDC4 transfected cos-7 cells.

[3H]-LSD showed saturable specific binding to a single

p o p u l a t i o n of sites with nanomolar affinity.

The affinities of

various 5-HT receptor drugs for the receptor closely matched the profile expected for a 5-HTID subtype 5-HT, 5-MeOT, LSD, affinities

for

sumatriptan and RU 24969, and low-intermediate

for (-) propranolol,

and ketanserin.

(16): namely high affinities

8-OH-DPAT, mesulergine,

ICS 205-930

As the 5-HTIB and 5-HTID receptors are closely related

(e.g. similar anatomical distribution)

compounds like (-) propranolol

or RU 24969 which are more selective for the 5-HTIB than for 5-HTID subtype were also tested and allowed us to further exclude the p o s s i b i l i t y that RDC4 could be a 5-HTIB receptor.

Furthermore,

5-HTIB

receptors have been found only in rodents whereas 5-HTID receptors have been found in pigeon,

cat, dog, human, bovine and other species

(16). Finally,

a significant correlation was found between the K i of a

v a r i e t y of compounds

for the RDC4 coded receptor and those reported

for the 5-HTID receptor.

All these data strongly suggest that RDC4

encodes a 5-HTID type of receptor. However,

although 5-HTID receptors have been reported to inhibit

adenylate cyclase,

RDC4 receptor was shown to be positively coupled to

adenylate cyclase.

One possible explanation for this discrepancy is

that this receptor is an adenylate cyclase activating subtype of the 5-HTID family.

The presence of two sub-populations of 5-HTID binding

sites has indeed been reported in porcine brain In the guinea-pig,

(17).

5-HTID sites are clearly negatively coupled to

adenylate cyclase in the substantia nigra, whereas in the c a u d a t e - p u t a m e n no consistent inhibition was found (16). Alternatively,

the same receptor could be linked to different

t r a n s d u c i n g mechanisms,

d e p e n d i n g on the G-proteins present. 1466

Vol. 180, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The 5-HTIA receptor has been shown to elicit distinct biochemical responses in different cellular systems (18). Similarly, the same TSH receptor which in dog thyroid cells is only activating adenylate cyclase, is also able to stimulate phospholipase C in human thyroid cells

(19). Whether our 5-HTID receptor can negatively regulate

adenylate cyclase will be shown in cloned permanently transfected cell lines. The identification of a 5-HTID receptor opens the way to the cloning of the human receptor and further putative subtypes. As this receptor and sumatriptan seem to play a role in the pathophysiology and treatment of migraine, the availability of a cloned 5-HTID receptor could be helpful in pharmacology.

ACKNOWLEDGMENTS

The authors would like to thank Dr A.P. Czernilofsky and J. Leysen for helpful discussions. This text presents results initiated by Prime Minister's Office, Science Policy Programming, the "Fonds de la Recherche Scientifique M~dicale", "Bender and Co (Boehringer Ingelheim), Vienna, Austria" and "A.P.M.O." C. Maenhaut and F. Libert are "Aspirant" F.N.R.S.

REFERENCES

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