High affinity receptors for atrial natriuretic factor in PC12 cells

Vol. 156, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 78-85

October 14, 1988

HIGH AFFINITY

RECEPTORS FOR A T R I A L N A T R I U R E T I C FACTOR IN PC12 CELLS

A. R a t h i n a v e l u

and G.E.

Isom

D e p a r t m e n t of P h a r m a c o l o g y and T o x i c o l o g y School of Pharmacy and Pharmacal Sciences Purdue University, West Lafayette, IN 47907

Received August 1, 1988

S p e c i f i c receptors for atrial n a t r i u r e t i c factor (ANF) are characterized on PC12 cells (rat p h e o c h r o m o c y t o m a cell line) . R a d i o i o d i n a t e d synthetic ANF (Rat, 8-33) b o u n d to a single class of high a f f i n i t y b i n d i n g sites w i t h the Kd v a l u e of 6.7 X i0 -I0 M. The Bmax value was 29 fmol/10 u cells and r e c e p t o r density was c a l c u l a t e d as 194,000 + 20,000/cell. P h o t o a f f i n i t y labelling of ANF receptor specifically labelled two protein bands with apparent m.wt of 70,000 and 130,000. When the cells were incubated with the labelled ligand at 37°C the I igand was internalized. The rate of i n t e r n a l i z a t i o n increased in the p r e s e n c e of increased ligand concentration. ANF receptors on PC12 cells are reported for the first time w h i c h would provide a unique model for study of ANF-receptor interaction. ® 1988Academic Press,

Inc.

The fluid

regulatory

volume

role

(1-2)

since

discovery

the

are

ANF

found

The

has

to

aldosterone

and

class

of

with

guanylate

cyclase

protein

with

and

adrenal serve

on

whereby

it elicits

adrenal

report

pheochromocytoma

affinity

its

the cell

(3)

and

activity,

for

ANF

78

for ANF

(6)

synthesis

and

a a

ANF

that

the of

zona glomerulosa

in p h e o c h r o m o c y t o m a effect

intracellular is not

receptors

is w i d e l y

adrenal

Inhibition

regulatory

the

on

suggest

for ANF.

effect(s) of

line which

0006-291X/88 $1.50 Copyright © 1988 by Academic Press,,Inc, All rights of reproduction in any form reserved.

domain

cells organ

the

yet

control

presence

hormone.

tissues

in the adrenal

confirms

function,

muscle

investigations

enzyme

receptors

medullary

and release

ANF

smooth

in

(4).

of c a t e c h o l a m i n e

by

hormone

receptor

as the target

synthesis

(8)

a

(ANF)

peptidic

coexist

GMP

Factor

numerous

in v a r i o u s

(7) and inhibition cells

new

of

localized

of high

glands

we

this

presence (5)

focus

are

for cyclic

adrenal

study

of

Natriuretic

natriuresis the

domain

cortex

tumor

been

receptors

bifunctional catalytic

Atrial

homeostasis,

relaxation

Specific

of

used

this

mechanisms

known. in

of

PC12

for the

In this cells, study

a of

Vol. 156, No. 1, 1988

catecholamines a unique with

model

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

biosynthesis to study

catecholamine

MATERIALS

and secretion.

the

synthesis

interaction

This

of ANF

cell

line offers

receptor

dynamics

and release.

AND METHODS

Materials 125 S y n t h e t i c rat Atrial Natriuretic Factor (ANF 8-33) and [ I]-ANF (Rat, 8-33, specific activity 1200-1500 Ci/mmole) were purchased from Peninsula Laboratories Inc. (Belmont,CA). E l e c t r o p h o r e s i s m a t e r i a l s were p u r c h a s e d from B e t h e s d a R e s e a r c h Laboratories and bis(Sulfosuccinimidyl) suberate was obtained from Pierce Chemical Company. All other c h e m i c a l s u s e d in these e x p e r i m e n t s w e r e purity grade. Cell

Culture PC12 cells were obtained from American Type Culture C o l l e c t i o n (CRL NO.1721) and m a i n t a i n e d in RPMI m e d i u m c o n t a i n i n g 10% heat inactivated horse serum, 5% fetal b o v i n e serum, 50 units/ml penicillin and 50 #g/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Just p r i o r to the binding assay, the cells were h a r v e s t e d from tissue culture flasks and w a s h e d three times with serum free RPMI medium c o n t a i n i n g 0.2% BSA. Binding Experiments ANF b i n d i n ~ assays in intact PC12 cells was p e r f o r m e d by incubating 5x10 ~ cells w i t h [125I]-ANF in the b i n d i n g m e d i u m (RPMI m e d i u m c o n t a i n i n g 0.2% BSA) in a total v o l u m e of 200 ~i, at 0°C for 2 hrs. All assays were p e r f o r m e d in p o l y s t y r e n e tubes. At the end of incubation the bound and free r a d i o l i g a n d were s e p a r a t e d by rapid filtration using GF/C glass fibre filters t r e a t e d w i t h 0.02% Tween-20 in PBS for 2 hrs. The filters were w a s h e d rapidly w i t h an additional i0 ml of ice cold 0.15 M NaCl and c o u n t e d in a gamma s c i n t i l l a t i o n counter. I n t e r n a l i z a t i o n of A N F - r e c e p t o r s PC12 cells were w a s h e d in p r e w a r m e d b i n d i n g m e d i u m three times, loosely pelleted by centrifugation at 300 X g and incubated w i t h [125I]-ANF at 37°C. At the end of incubation p e r i o d the tubes were drawn from the incubator and p l a c e d on ice. The cells were w a s h e d by adding ice cold 0.15 M NaCI and c e n t r i f u g e d at 300 X g for 2 min. at 0°C. ANF b o u n d to the cell surface was d e t e r m i n e d by e x t r a c t i o n of the intact cells with 0.5 M NaCl, 0.2 M acetic acid (9). The cells were s o l u b i l i z e d in 1 N NaOH and the radioactivity associated with the cells was d e t e r m i n e d w h i c h r e p r e s e n t e d the i n t e r n a l i z e d ligand. A f f i n i t y C r o s s - l i n k i n q Studies C o n f l u e n t cells from 150 cm 2 flasks w e r e w a s h e d w i t h serum free RPMI m e d i u m and h o m o g e n i z e d in the ice cold b i n d i n g m e d i u m c o n t a i n i n g 0.25 M sucrose, 0.4 m M p h e n y m e t h y l s u l f o n y l fluoride, 1.0 mg/ml bacitracin, 20 #g/ml each l e u p e p t i n and aprotinin. The h o m o g e n a t e was c e n t r i f u g e d at 1,500 x g for i0 min. and the s u p e r n a t a n t was c e n t r i f u g e d at i00,000 x g for 30 min. The p e l l e t was r e s u s p e n d e d in the b i n d i n g m e d i u m and c e n t r i f u g e d again at i00,000 x g for 30 min. to remove sucrose. The w a s h i n g step was repeated twice and the final pellet obtained was incubated w i t h the labelled ligand in a total v o l u m e of 200 #I for 90 min at 37°C. 200 #i of 0.2 m M bis(sulfosuccinimidyl) suberate in DMSO was added and the incubation was c o n t i n u e d for

79

Vol. 156, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

another 30 min. At the end t r a n s f e r r e d to ice, c o l d b i n d i n g at i 0 0 , 0 0 0 x g for 30 min. electrophoresed a c c o r d i n g to t h e gel. T h e g e l s w e r e d r i e d and - 7 0 ° C for 30 days.

of incubation the tubes were m e d i u m w a s a d d e d and c e n t r i f u g e d The p e l l e t was solubilized and m e t h o d of L a e m m l i (i0) u s i n g 10% e x p o s e d to K o d a k X - O M A T film at

RESULTS ANF

Receptor PC12

ANF. a

show high

Scatchard

single

constant M

Bindinq

cells

(29

sites

class (Kd)

[125I-ANF

of

yielded

binding

for A N F w a s

fmol/105 are

analysis

cells).

calculated was

was

reached

ANF

displaced

the

specificity

a

a linear

sites;

on this

194,000

dependent

120

almost

saturable

binding

plot

the

for

(fig.l)

apparent

±

the

number Also

process

of

of r e c e p t o r

and

2 #M

labelled

indicating

1.94

20,000/cell.

min.(fig.2).

80%

data

of b i n d i n g

equilibrium

binding

X i0 -I0

binding

concentration

ligand

[125I]-

dissociation

6.7 X i0 -I0 M and B m a x w a s

Based as

time

within

affinity,

of

binding of

cold

indicating

binding.

co i 0 x 0

20

Specific 6

E a.

16

!

12

~

8 0 ~ 0

(D

4

/ Jo-3°Lou 0.4

0

0.8

(1251)-

1.2

1.6

Q

Non-Specific

2

60

120

180

MINUTES

A N F nM

Fig. l: Saturable b i n ~ n g of [125I]-ANF (rat, 8-33 ) to PC12 cells. The cells (5x10 cells/tube) were incubated at 0°C with various concentrations of [125I]-ANF for 2 hrs. in RPMI medium containing 0.2% BSA. Specific binding is the difference between total binding and the non-specific binding in the presence of 2 ~M unlabelled ANF (rat,8-33). (Inset) S c a t ~ a r d plot of the binding data. The calculated Kd is 6.7 x 10-~VM and Bmax is 29 fmol/105cells. Binding of [125I]-ANF (rat,8-33) to PC12 cells as a Fig.2 : function of time at OoC with labelled ANF. Non-specific binding was determined in the presence of 2 #M unlabelled ANF (rat, 833)

.

80

240

Vol. 156, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(D

0 x

E

Q.

o Q

30

2N

24

Z2.4

tU N m

3.2

.,I

< Z

IZ m

z

u.

.~1.6

16

Z

o

,<

< I

8

~--

"0,8

04

i

40

Q

i

i

80

120

4

MINUTES C.

8

Q

12

MINUTES

Time course of [125I]-ANF internalization by PC12 cells The cells were incubated for 120 min. with 2 nM [125I]-

ANF. Fig.4: Internalization with 0.25 [ • ] n M ANF concentrations at 37°C.

Internalization When

of 1-125

the

cells

internalization inability

of

associated rapid

at

The

reaching was

the

to

also

0.25

was

0.25

concentration

determined

surface-bound This

plot

calculated and t h e

higher

by

plotting

for

as a

ANF rate

of A N F

concentrations

a

with

the

constant

of

ANF

The was 81

at

a

to

increase

concomitant

When

2

nM

of

than

incubation

in

was

to

(ll).

Ke.

The

0.045

m i n -I

m i n -I

the

with

the (Ke)

ligand

of

as

surface

constant

slope

0.092

of

ANF

The

rate

concentrations

increased

(fig.6).

cell more

concentrations

internalized

time

with

the

was

2 nM concentration

of

curve

by

(fig.3).

(fig.4).

of

37°C,

the

ligand

rapid

endocytic

ratio

nM

min.

more

function

0.25

20

different

the

remove

the

two

at

indicated

of

was

The

linear at

at

concentration

(fig.5).

ligand

endocytic

concentrations

nM

the highest

produced Ke

at

was

completely

maximum

observed

binding

was

a

to

[125I]-ANF

This

binding

internalization

the

with

observed. acid

[125I]-ANF,

nM

was

NaCl/acetic

internalization

compared

incubated

radioactivity. 37°C,

O ] [125I]-

ANF

were

of A N F

and 2 n M [

Ke an

at

2 rum

value

at

increased

Vol. 156, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6

0.6

==

~0.4

o

~0 ,

0.2

4

Q

8

12

4

MINUTES

Q

i

|

8

12

MINUTES

Fig.5: Surface binding of [125I]-ANF on PCI2 cells at 37°C with 0.25 nM [ • ] and 2 nM [ O ] labelled ANF for different time intervals. Fi~ In/Sur a ~ y s i s in PC12 cells at 37°C with 0.25 n M [ • ] and 2 n M [ O ] [ I]-ANF. The ratio of radioactivity associated with the interior to the surface of the cell (In/Sur) was determined. The slope of this curve yielded the endocytic rate constant Ke.

surface was

binding,

apparently

Affinity

thus

Labellinq

The

labelling

coupled

components

of

proteins

the

polyacrylamide

PC12

gel

In

proteins

was

labelling

of

indicated

the

in

internalization

plasma of

and

under

of 2 #M c o l d A N F

other

almost

fractions

of A N F

in t h e p l a s m a

to

membrane.

70,000

inhibited

specificity

bis(sulfosuccinimidyl)

specifically

m.wt

the presence

proteins

using

electrophoresis

(fig.7).

m.wt

reaction

cell

at

of

specific

increase

occupancy.

[125I]-ANF

migrated

these

observed

of A N F R e c e p t o r s

affinity

suberate

the

d u e to r e c e p t o r

protein

The

labelled

130,000

reducing the

with

on

SDS

condition

labelling

completely,

remained

binding

two

of b o t h

while

non-

unaltered. 70,000

and

This 130,000

membrane.

DISCUSSION A ANF

great

on

deal

adrenal

aldosterone glomerulosa has been (12), cells

of

synthesis (7).

adrenal

functionally

and

in a d r e n a l

medullary This

by ANF

focused

the

of h i g h

cortex (6) that

on

the

polypeptide

release

cells

suggests

regulated

has

since

The presence

reported

(8).

attention

responses,

from

the

affinity

(5), and

adrenal in h u m a n

influence

basal

adrenal

zona

receptors zona

for ANF

glomerulosa

pheochromocytoma

the

adrenal

glands

at v a r i o u s

levels.

Recent

82

of

inhibits

may

be

evidence

Vol. 156, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

130,000 70,000

A

B

Fig.7: Specificity of [125I]-ANF cross linking to ANF receptors on PC12 cells. The dried gel was exposed to KoCh% X-OMAT film at -70°C for 30 days. Specific binding of [ I]-ANF to the receptors in plasma membrane (lane A). Non-specific binding in the presence of 2 ~M unlabelled ANF (lane B).

of A N F the

secretion

adrenals

actions

may

present

on PC12

have

interesting

model

specific,

rapid,

indicates

that

in

mediating

the

affinity

receptors

for

(Kd)

previously

to

cyclase

elevation

of

the

cGMP

vascular

(18).

that

the

functionally

for

adrenal

smooth high

which

was

intracellular elevation

smooth

cells

Bmax

possess

guanylate

muscle

particulate coupled

the

cells

cells

levels

of

activated (16). in

(14)

of ANF

cells. 83

the

It

This

receptors

the

rat

(14)

cells

have

guanylate

form

is

similar

cells

in

of the

(17),

endothelial

cyclase as

in

aorta

aortic

on PC12

receptors,

values

resulting

and

was

cortex

response

bovine

of

dissociation to

particulate

rabbit

and

sites.

PC12

ANF

of

many

renal

(15).

by

aspects

The

(6),

as an

fulfills

comparable

medullary

of A N F form

to

are

muscle

served

functional

saturable.

observed

has

recognition

values

cGMP

cells

Demonstration

the to PC12

and

and

vascular

line w h i c h

receptor

reversible

reported

reported

of A N F

specific

value

to

(13)

roles

high

cell

exploring

Binding

for

cultured

demonstrate

for

cells.

criteria

been

cells

complicated

a transformed

constant

and

medullary

more

results

cells,

chromaffin the

adrenal

of ANF.

The ANF

by

suggest may

many

be other

VoI. 156, No. 1, 1988

Following receptors

on

labelled

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

covalent plasma

membranes,

and migrated

conditions. reported

These

in

endothelial

bovine cells,

fibroblast

coupling

at m.wt

70,000

high

reducible

and

receptors

are

activity,

non-reducible reported

ligand

were

and 130,000

to

smooth

two

of

(5), cells

weight

to ANF

specifically

under

classes

cells

muscle

molecular

from the bovine adrenocortical cyclase

proteins

adrenocortical

The

guanylate

two

correspond aortic

(19).

of radiolabelled

reducing receptors

bovine and

aortic

human

protein,

lung

isolated

cells showed both ANF binding and

which

is also

forms.

not

The

coupled

to

believed low

to exist

molecular

particulate

in

weight

guanylate

cyclase. PC12 cells

appear to possess

linked to the particulate

two classes

form of guanylate

of receptors,

cyclase,

one

since the

high molecular weight receptor was not completely reduced to the low molecular weight protein, fractions,

reducible

forms of high molecular weight receptor proteins may be

present

in PC12

interesting with

the than

cells.

phenomenon, ligand

internalization other

it

is

as indicated by the presence of two

labelled

speculated

The

the

concentration. process

in

reducible

internalization

since

receptor-mediated

both

these

process

internalization It

is

cells

endocytosis,

perform

such

varies

that

the

functions

as transmembrane

signal transduction or diminishing the cell responsiveness ligand by removing the cell surface receptors

non-

is another rate

speculated may

and

to the

(20).

REFERENCES I. 2.

3. 4. 5. 6. 7.

Cantin, M., and Genest, J. (1985) Endocrin. Rev., 6, 107126. Ingami, T., Misono, K.S., Grammer, R.T., Fukumi, H., Maki, K., Tanaka, I., McKenzie, J.C., Tajayangi, R., Pandey, K.N., and Parmentier, M. (1985) Clin. Exp. Hypertens. Part A theory Pract.,A7, 851-866. Roubert, P., Lonchampt, M.O., Chabrier, P.E., Plas, P., Goulin, J., and Braquet, P. (1987) Biochem. Biophys. Res. Commun., 147, 957-963. Kuno, T., Anderson, J.W., Kamidsaki, Y., Waldman, S.W., Chang, L.Y., Saheki, S., Leitman, D.C., Nakane, M. and Murad, F. (1986) J. Biol. Chem., 261, 5817-5823. Takayanagi, R., Ingami,T., snajdar, R.M., Imada, T., Tamura,M., and Misono, K.S. (1987) J. Biol. Chem., 262, 12104-12113. Heisler, S. and Morrier, E. (1988) Biochem. Biophys. Res. Commun., 150, 781-787. Goodfriend, T.I., Elliot, M.E., Atlas, S.A. (1984) Life Sci., 35, 1675-1682. 84

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