BIOCHEMICAL

Vol. 183, No. 2, 1992

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

March 16, 1992

RAT

LUNG

Centre

Received

POSSESSES

THE

M.

Agarwal

Universitaire

January

20,

K.

MINERALOCORTICOID and

M.

des Cordeliers, 75270 Paris Cedex

405-410

RECEPTOR

Mirshahi

15 rue de 1’Ecole 06, France

de Medecine

1992

SUMMARY: Lung cytosol from male, adrenalectomized rats was screened for the mineralocorticoid receptor (MCR) by a polyclonal antiserum raised in the rabbit against rat renal antigen. Western blot analysis revealed a single 98 kDa band, like the MCR purified biochemically. The MCR could also be photolabelled for the first time by H-R 5020 in this very 98 kDa region that was displyed by RU 26752 specific to MCR. Immune IgG was able to precipitate the MCR- H-RU 26752 complex, and to displace the same to high molecular weight regions during gel permeation chromatography on Sephacryl columns. Thus, CICR mediated actions need to be redefined. Furthermore, the technique of photochemical labelling forms a novel tool to assess MCR specificity, and to 0 1992 Academic Press. 1°C. dissect its structure and function.

INTRODUCTION: receptors

Steroid

that

trans-activate

(l-3).

The

control

of mineralocorticoids

etic

regulation

antagonists

nical

receptor

regulate

sodium

modulation

this

for

classical transport the

1abeIling

where

at epithelial

detection,

Since

the

in the

and

developed

and

the

presence

organs

believed

(11))

target

tissue, to effects

directly

generation

of the

These

data

that

and

organs

the that

of

technique here question

to promote

such

to

we screened

The

merely

mineralo-

that

us to demonstrate

cytosol.

biocli-

contribute

development

permits

of synth-

of the

enzyme

the

specific

may indirectly

were

extend

is under

for

in various lung

types

a number

or Mineralocortin.

pulmonary

mineralocorticoids

(4-6)

converting

RICR,

in rat

mammal

The

to specific of cell

in the

been

laboratory,

should

array

demonstrated

the

surfaces

in a whole

(7-10).

of MCR,

, in our

action

have

an Angiotensin

of mineraloreceptor view

been (4-6).

against

by binding

as aldosterone

actions

presence

antiserum

mineralocorticoid

escaped

has

via

possible

of photoaffinity existence

mediated

to act

balance

ZK 91587)

homeostasis

hydrosodic

a polyclonal

hydrosodic

26752,

(MCR)

believed

expression

such

of MCR

corticoid

are

gene

of the

(RU

dissection

tissue

hormones

the the

sodium

definition

of

had

hitherto.

405

All

Copyright Q 1992 rights of reproductim

0006-191x/92 $1.50 by Academic Press, [TIC. in any form resented.

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183,

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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MATERIALS AND METHODS : The activated MCR was purifiq from rat kidney by our original procedures (12,13) consisting of: receptorII-RU 26752 equilibration, activation, phosphocellulose chromatography, DNA cellulose elution, and lyophilization, in that order. The lyophilized MCR (150 Bg protein) was mixed with Freund’s adjuvant and injected into fawn, Burgundy rabbits, followed by a booster shot three weeks later. Anti-MCR titers were quantitated by the Enzyme-linked immunoabsorbent assay (ELISA) according to the standard protocol described in detail recently (14). The five week bleed was used to separate the IgG fraction on a DEAE-Trisacryl M (lot 5208, IBF, France) column (14). Lung cytosol from adrenalectomized, male, Wistar rats (Iffa Credo, France) was prepared in phosphate buffered saline (PBS) containing (per 1) 0.25 g potassium dihydrogen phosphate, 1.38 g disodium phosphate, 0.25 g KCl, 9 g NaCl, and 0.01% sodium azide. For Western blots, the cytosol was denatured in 220 mM Tris-HCl, pH 6.8, 2% SDS, 15% glycerol, and electrophoresed for 3-4 h on 15% acrylamide gels in Tris-glycine-SDS (13 g + 14.4 g + 1 g/l water). Molecular weight markers (Biorad) were run simultaneously and stained by Coomassie brilliant blue (12-14). The proteins separated by SDS-PAGE were electrotransferred to nitrocellulose membranes (Millipore) for 1 h at 5mAmp in Tris-glycinemethanol (1.5 g + 7.2 g + 100 ml/l), blocked for 1 h with 15% non fat dry milk, incubated with rabbit anti-MCR antiserum for 2 h at 4O C, and finally saturated with sheep anti-rabbit biotinylated antibody for 90 min, followed by 90 min in presence of streptavidin biotinylated horseradish peroxidase complex. The blots were developed in dark with hydrogen peroxide-4-choloro-l-naphthol-methanol (0.01%:0.05%:10%) and dried. For photochemistry, lung cytosol was saturated with 50 nM 3 H-R 5020 alone or in presence of one hundredfold excess of RU 26752, that is specific to MCR, for 2 h at 4OC. This was followed by irradiation at l: 10000

titers

immunoabsorbent

of MCR-

Vol.

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BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

RESEARCH

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2

a

b

kDa

1.5

d

97 $1 z

67-

g 430.5

310

0 1

Dilution 36

12

Figure 1. Blood 150 ug of ag of the

106

324 X 100e2

02

972

14-

Quantitation of the rabbit antibody to Mineralocortin by ELISA. samples were analyzed three weeks after the initial injection of rat renal MCR (I), and three weeks after a booster shot of 140 same preparation (a ) ; control, nonimmune serum ( 0).

Figure 2. Immunophotochemical demonstration of mineralocortin in rat lung. Standard molecular weight markers (Biorad), rat pulmonary cytosol after SDS-PAGE (a), Western blot of lung cytosol (b), autofluorography of lung cytosol photolabelled with tritiated R 5020 alone (d) or in presence of excess RU 26752 (c). For further details see text.

assay

(ELISA)

purified

just

from

Three

rat

weeks

such

that

cytosol

in Fig. was

estimate

targets,

are

devoid

Since chemical

labelling

the

in the Technical

a specific

with

region

specificity

a single

Western

the

MCR

renal

shown)

for

of this

lung

an excess

titers

of RU 38486.

increased of the

of about

98 kDa

when

agreement

human

kidney

(15),

was

wanting

This

organs

are

not

even

further

antiserum.

good

(12-14).

these

and

with

rat

the

lung

theoretical

and

with

when

the

liver

or

nineralocorticoid

(12-14).

promegestone

when

in from

antigen since

receptor

blot,

150 l.~g of protein

dilution

band

cloned

of NlCR

with

the

at >1:32000

photoprobe

progesterone

98 kDa

140 ug MCR

show

the

rat

(not

and

b)

for

used

immunization of RU 26752

positive

by

purified

was

with

still

2 (lane

of 107 kDa

thymus

label

was

after

in presence

a booster

analyzed

biochemically

weeks

kidney

after

ELISA

Data

three

the

MCR is wanting,

that (3).

Data

cytosol

procedure

has

been

in Fig.

successfully 2 (lane

was photolabelled was 407

evident

we attempted

from

d) with the

used show 3H-R absence

phototo photo-

a single 5020. of this

band

Vol.

183, No. 2, 1992

BIOCHEMICAL

band with either the radioligand

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

alone, or when MCR-specific

allowed to compete with 3H-promegestone (Fig.

2 lane cl.

RU 26752 was

Pretreatment

of lung

cytosol with the immune serum, too, abolished the 98 kDa band revealed by photochemical fluorography

(similar to lane c) .

Data in Fig. 3 show that immune IgG was able to precipitate MC8 complexes. The precipitation

3H-RU 26752-

was limited to no more than 35% of the total

cellular NCR, even with increased amounts of immune IgG, as with other classes of steroid hormone receptors was required

(16-18).

for optimal resolution,

to the native heterooligomer in vlvo

Furthermore,

the presence of high salt

suggesting limited access of the antibody (14,16-18).

Data in Fig. 4 show that incubation

with immune IgG increased the molecular

mass of rat pulmonary MCR. In presence of nonimmune IgG, one sharp peak of about 840 kDa was followed by another component in the 44 kDa region, ting degradation

and polymerization

during the chromatographic

04 Figure

3.

Figure

4.

25

procedure

50 FRACTION

of

rat

pulmonary

mineralocortin

by

immune

IgG.

Rat lung cytosol labelled with 20 nIv1 311-RU 26752 was incubated with 500 tig of either the immune (e) or non-immune (0) IgG and thereafter passed through a Sephacryl S-300-IIR column, as described in the text. Approximate mass in kilodaltons has been indicated above each peak.

408

(14).

15

NUMBER

Immunoprecipitation of rat pulmonary mineralocorticoid receptor by immune IgG. Rat lung cytosol radiolabelled with 20 r&l 311-RU 26752 alone plus 5 ug immune IgG (H); cytosol labelled as above in presence of 1000 fold excess of radioinert RU 26752 plus nonimmune IgG (Cl 1. The values represent the average of three separate determinations. Macroaggregation

sugges-

Vol.

183,

No.

In presence 70 kDa,

of anti-MCR

preceded

gate

whose

and

Fab

high

corticoid

as with

from

the

targets cross

specific

RU 26752, studies

delineating

the

the

manner

shifted

an even

accurately. with

to about

larger

Thus,

aggre-

both

the pulmonary

IgG

MCR in the

although

the

latter

did

lung

is endowed

with

the

mineralo-

that

appears

in the

pulmonary estro-progestative interesting for

mammalian

steroid

not

the

insights. mineralocorticoid

MCR

kidney

(10,12,13,19).

Photo-

that

could

be displaced

by MCR-

that

binding

believed

could

be exploited

in

domain

of this

receptor,

complexes

bind

to DNA

kidney)

Receptor

androgen

Unconventional

(23), and

sodium that

significance

unknown. and

to promote

(heart,

physiological

(22),

to the

and for

genome.

targets

remain

identical

the

MCR-steroid

generally

in classical

tissue

and

of identity

in which

are

Therefore,

rat

promegestone,

for

surfaces lung.

heart

proof

of the

in the

as the

with

Mineralocorticoids

be anticipated

peak

and

(16-18))

that

is a formal

trans-activation

provided

to complex

Mineralocortin,

such

linking

to understand

21),

be estimated

receptors

foregoing

protein,

chemical

epithelial

other

region,

COMMUNICATIONS

estimates.

receptor

in classical

able

RESEARCH

of the lighter

560 kDa

not

are

BIOPHYSICAL

position

in the

could

thereof

buffer,

It is clear

future

mass

AND

the

a hump

molecular

size

IgG,

by

fragments

salt

provide

BIOCHEMICAL

2, 1992

and

is apparently the

mediated

absorption

cell

types

glucocorticoid

action

in the

lung

novel

mechanisms

across wanting involved (20,

has

already

may

similarly

action.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

M. Beato, Faseb J., 5 2044-2051 (1991). W. Wahli and E. Martrnez, Faseb J., 5 2243-2249 (1991). M. K. Agarwal, Die Natur., 77 170-l% (1990). G. VJ. G. Sharp and A. LeafFPhysiol. Rev., 46 593-633 (1966). J. W. Conn, J. Am. Med. Assoc. 183 775-781 fl955). J. Crabbe, J. Clin. Invest., 40 2103-2108 (1961). M. K. Agarwal and G. Lazar,xenal Physiol. Biochem., 14 217-223 (1991). M. Kalimi, J. Opoku, M. K. Agarwal and K. Corley, AmTJ. Physiol., 258 E737-739 (1990). J. Opoku, M. Kalimi, M. K. Agarwal and D. Qureshi, Am. J. Physiol., 260 E269-E271 (1991). M. K. Agarwal and M. Kalimi, Biochim. Biophys. Acta, 964 105-112 (1988). K. K. F. Ng and J. R. Vane, Nature, 216 762-763 (1967). G. Lazar, M. Pagan0 and M. K. Agarwal, Biochim. Biophys. Acta, 1033, 41-48 (1990). G. Lazar, M. Pagan0 and M. K. Agarwal, Int. J. Biochem., 22 621-630 (1990). 409

Vol.

183, No. 2, 1992

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

14. M. Mirshahi, M. Pagano, A. Mirshahi and Ill. K. Agarwal, Biochim. Biophys. Acta (in press). 15. J. L. Arriza, C. Weinberger, G. Cerelli, T. RI. Glaser, B. L. Handelin D. E. Houseman and R. Evans, Science, 237 268-275 (1987). 16. G. L. Greene, L. E. Closs, H. Fleming, E. R. DeSombre and E. V. Jensen, Proc. Nat. Acad. Sci. (U.S.), 74 3681-3685 (1977). J. Krco and D. 0. Toft, 17. W. P. Sullivan, T. G. Beito, J. Proper,C. Endocrinol. , 119 1549-1557 (1986). 18. A. Traish, Rxttinger, N. Kim, A. M. Rothstein and H. H. Wotiz, Steroids, 55 196-208 (1990). 19. M. K. Agarwal, FEBS Letts., 85 l-8 (1978). 20. G. Giannopoulos, J. Biol. Chez, 250 2896-2903 (1975). 21. M. K. Agarwal and M. Philippe, Blochim. Biophys. Acta, 500 42-48 (1977). 22. P. T. Cagle, D. Mody and M. R. Schwartz, Can. Res., 50 6632-6635 (1990). 23. W. T. Morishige and C. A. Uetake, Endocrinol., 102 18277837 (1978).

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