Calmodulin regulation of cholinergic muscarinic receptor: Effects of calcium and phosphorylating states

Vol.

133,

No. 3, 1985

December

31,

BIOCHEMICAL

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RESEARCH

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1985

1193-l

200

CALMODULIN REGULATION OF CHOLINERGIC MUSCARINIC RECEPTOR: EFFECTS OF CALCIUM AND PHOSPHORYLATING STATES Andrew

K.S.

Ho" and Jerry

H. Wang

Cell Regulation Group University of Calgary Calgary, Alberta, CANADA *Department of Basic Sciences University of Illinois College of Medicine at Peoria Peoria, Illinois Received

November

5, 1985

Calmodulin (C&i) regulation of cholinergic muscarinic receptor was investigated using synaptic membrane isolated from rat brains and [3H]-QNB as a binding ligand. CaM exerts a biphasic effect on receptor binding showing both a Ca2+-dependent receptor loss and an increase depending Calcineurin, a &M-dependent on the state of membrane phosphorylation. protein phosphatase, mimicked the stimulatory effect of CaM in a dosedependent manner. &M-antagonists, W-7 and TFP reversed the stimulatory phosphorylation and dephos horylaeffect by CaM. A mechanism of protein tion of the cholinergic muscarinic receptors regulated by CaM-Ca 2P was proposed. Q 1985 Academic Press, Inc. Burgoyne muscarinic

(1)

demonstrated

receptor

der phosphorylating antagonized However,

hibits

conditions

and in the presence

kinase

communication, using

a different

phosphorylating of [3H]-QNB

[3H]-QNB

states

phosphatase, dephosphorylation

we have

effect

binding (3,4)

phosphatase

of the

was mimicked suggesting

activities

(2).

loss

the effects Our results and increase

membranes.

by calcineurin, role

of muscarinic

This

loss

effect

unwas

showed depending

a CaM-dependent of protein

by the

of CaM and Ca2+

The CaM-induced

that

CaM ex-

on the activation protein

phosphorylation-

receptor.

ABBREVIATIONS USED: EGTA, Ethyleneglycol-bis-(S-aminoethylether) acetic acid; W-7, N-(6-aminohexyl)-5-chloro-1-chloro-1-napthalene PNPP, para-nitro-phenylphosphate; TFP, Trifluoperazine.

1193

([3H]-QNB)

and 2-deoxyadenosine. of receptor

a possible

in the regulation

of CaM.

a reversal

as a ligand.

synaptic

to the cholinergic

benzilate

investigated

on receptor

binding

adenosine

inhibitors,

to demonstrate

of exogenous

on AChR binding

specific

L-[3H]-quinuclidinyl

he was unable

In this

in the

antagonist

by protein

manipulation

a loss

N,N'tetrasulfonamide;

0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

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MATERIALS -~ AND METHODS Sources: L-[3H]-QNB, spec. act. 30.2 Ci/ti (New England Nuclear N-(6-aminohexyl)-5-chloro-1-napthalene cyclic AMP, CaM antagonists, mide, (Sigma), DFAE, Sepharose 48 from Pharmacia (Uppsala).

Corp., Mass.), sulfona-

The synaptic membranes from whole brains of rat were prepared according to Jones and Matus (5). all procedures were carried Except otherwise specified, out at 4°C. The synaptic membranes were washed once with approximately 10 volumes of Tris buffer (50 mM) pH 7.5, centrifuged again and the pellet was suspended in Tris buffer and used either fresh or stored at -7O'C. For incubation under phosphorylating conditions, membranes were resuspended in buffer (A): containing Tris (50 mM) pH 7.5, MgAc (1 mM), ATP (1 mM); CAMP (50 FM), CaC12 (50 PM), CaM (20 or 40 ug/ml). The control membranes were treated similarly with Tris buffer. The mixtures, after incubation at 30°C for 10 min., were centrifuged at 25,000 x g, washed and pelleted as before. Calmodulin and calcineurin were purified to homogeneity from bovine brain by the method of Sharma et al. (6). The phosphatase activity of calcineurin was activated by preincubation with 1 mM Ni2+ for 60 min as previously described (7). To study the effects of EGTA treatment on receptor binding, the phosphorylated membrane preparation was divided into two halves. One half was incubated at 30°C with 10 vols of EGTA (1 mM) for 10 min, washed twice with 10 vols of Tris buffer, pelleted at 25,000 x g to remove all the EGTA and membrane CaM. The other half was similarly treated with Tris buffer but without EGTA. The CaMCa2+ effect on receptor binding was again determined using both membrane preparations and assayed in Tris-buffer only. Muscarinic receptor assays using [PHI-QNB were carried out with synaptic membrane (40-100 ug protein) in 0.1 ml either in Tris-buffer (50 mM, pH 7.5) or in the presence of appropriate amounts of added substances (CaM, calcineurin, Ca2+, CAMP, ATP, Mg2+>. After 10 min preincubation, the binding assays were initiated with the addition of 10 ul of [3~]-~~~ (3.2 nM final concentration). The assay mixtures were incubated in duplicates with and without 10 nM atropine sulphate at 30°C for 30 min in a Dubnoff water bath with constant shaking. The reactions were terminated by addition of ice-cold buffer (3 ml) filtered under vacuum through Whatman GF/B glass fibre filter discs and washed three times each with 3 ml buffer. Specifically bound [3~]-~~~ was calculated from the difference between the mean binding in the absence and presence of atropine and the data expressed as fmoles bound per milligram protein which was determined by the method of Lowry et al. (8) using bovine serum albumin as standard. RESULTS AND DISCUSSION ~The results exert

significant

depending

were

loss carried

following out

showed

clearly

on the cholinergic states

reports

Using

different (1)

membrane

binding and the levels

conditions,

on the muscarinic

of ATP (1 mM), cyclic

membrane.

we receptor

Our assays

AMP (50 PM), Mg2'

The down-regulation

1194

CaM and Ca2+

receptor

assay

of the synaptic

Following

both

muscarinic

by Burgoyne

and Ca2+ (50 nM). by EGTA.

that

of the synaptic

phosphorylation

in the presence

and abolished

study

CaM and Ca2+.

the earlier

(1 mM), CaM (20 ug/ml) dependent

this

phosphorylating

or added

confirmed

binding

in

alteration

on the

of endogenous have

obtained

EGTA treatment

by CaM was doseof the

phosphory-

Vol.

133,

No.

BIOCHEMICAL

3, 1985

AND

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8

-Log

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4

CCa”lf

Graphs showing the negative log of free [Ca2+]f vs the specific QNB binding expressed as % of control. Synaptic membrane wai phosphorylated with Buffer A, CeM (20 ug/ml) and Ca2+ (50 nM) for 1 0 min. washed and resuspended in Tris-buffer. Assay mixture each containing synaptic membrane (60 ug protein approximately) CeM (20 ug/ml), CeM (20 rig/ml) plus EGTA (1 mM) or control vith no addition, were added various concentrations of (Ca2+)f, preincubated for 10 min before the addition of [3H]-QNB, and incubated for 30 min as described in the method. Data were expressed as % of control; the assays containing no added [Ca2+] were used as 100%. The control mean values f SEM for specific activity were 630 ?: 41; 641 + 35 and 695 fmol/mg protein for the no addition, CaM, and CaM + EGTA respectively. (No. of experiments N f 4) for no addition and CaM N = 2 for EGTA + CeM. ?Fe [ H]

lated

membrane

and the removal

down-regulation, Fig.

CaM and Ca2+ produced

1 showed

pendent larly

the activation

on Ca 2' and this

by EGTA (Fig.

to

of

to note

binding

in the

non-phosphorylating preparation

first

that

fmol/mg

protein

assayed Following

a further

loss

CaM-Ca2+

same membrane

[3~]-~~~

and the

instead

receptor

by CaM (20 pg/ml)

of

binding.

was de-

of EGTA.

stimulation

with

buffer

The mean values in Tris

buffer,

EGTA wash, in receptor

can produce

preparation

For example,

phosphorylated

down-regulation.

showing

buffer,

by the addition

was observed

conditions.

typical

phorylation.

effect

Tris

of

binding

was abolished

with

Simi-

was abolished

2).

was interesting

[3H]-QNB

stimulation

[3H]-QNB

effect

a CaM dose-dependent

It

of EGTA by washing

data

under obtained

binding

and 560 fmoljmg

binding. 1195

from

However,

and

one membrane showed

sites protein

of 475 fmollmg

response

phosphorylating

A and CaM (20 rig/ml) of total

a value

a different

were after

protein

when assayed

a

633 phos-

was obtained, in the

Vol.

133,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

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80 40 pg /ml Figure 2. Graphs showing different concentrations of added CaM in the presence of either Ca2+ (0.1 mM) or EGTA (1 mM) on specific r31i]-QNB binding under experimental conditions described as in Fig. 1 and the method. 20

0

Calmodulln.

absence brane

of ATP and CAMP, addition was found

binding

loss

to have

has been

phosphorylation. (10 pg/ml

may also

which

consistently with

with

Ca *+ level

from

within

population

of

[3H]-QNB

binding

neuronal

tissue

somewhat

However, is

the

significantly

The level

with

may exist

but

cellular

than

that

than 1196

obtained

for

in

was of 20-25%.

those

in binding

of Ca 2+ and

the receptor

phosphorylated required for

and nonfor

other

activation CaM dependent

of CaM found used

vari-

may be attribuamount

used

CaM

the EGTA washed

a decrease

that

concentrations

greater

to

of stimulation

different

in both

prior

respectively.

the variations

of CaM (uM range) larger

level

to CaM-Ca 2+ produced

stimulation,

mem-

receptor

and in the range

response

for

the

binding

and the possibility

The amount is

[3H]-QNB

membrane

preparations

the membrane forms.

enzyme systems.

of

the membranes

within

phosphorylated

stimulation

The reason

only

mean values

and 697 fmol/mg

to slight

membrane

not

544 fmol/mg

the

EGTA pretreated

the initial

of Ca*+ (50 pM),

membranes,

lo-4M.

that

exceeded

the phosphorylated

no change

above

it

in

been phosphorylated.

than

to the crude

CaM present

produce

the control

results

effect

but

were

has not

less

able

table

restored,

In the presence

CaM-Ca*+

However,

a stimulatory

and 20 rig/ml)

membranes

of CaM and Ca*+ to this

in this

in the experiment.

Vol.

133,

No. 3, 1985

BIOCHEMICAL

Table 1. Effects

AND

BIOPHYSICAL

RESEARCH

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of C&i and Phosphatase(s) inhibitors on the Ca2+-CaM stimulation of specific [3~1-~~~ binding

Additions

Mean [3H]-QNB binding (X of control) 100 104 145 105 108.9 107.4 112.1 99.4 79.2 94.0 85.7

None

Ca2+ (0.1 mM) CaM (20 pg/ml) + Ca2+ (0.1 mM) W-7 (lo-5M) W-7 (10T5M) + Ca2+ + CaM TFP (lo-5M) TFP (lo-5M) + Ca2+ + CaM PNPP (5 mM) PNPP (5 mM) + CaM + Ca2+ EH2PO4 (lo-2M) KH2P04 + CaM + Ca2+

Synaptic membranes phosphorylated with ATP, CAMP, Mg2+, Ca2+ and CaM, washed and preincubated with various additions in Tris-buffer (50 mM), pH 7.5 for 10 min with or without atropine (10 M). [3Hl-QN13 was added and incubated for 30 min at 30°C. The mean specific [ YHI-QNB binding was expressed as percentage of the control value (655 fmol/gm protein). Data shown are the means from three or more experiments with variations within 5%. To further binding, that

W-7 (9)

activation

of

excessive

[3H]-QNB

not

shown).

the

stimulation

with

densities

up-regulation

activated

Results

antagonism

system

(10 ug/ml) inhibitors,

obtained

without

(Table

1).

because

it

known

receptor showed

to the CaM-induced

levels

activity

in this

phosphatase

producing Compound lowered

to antagonize

48180 the basal

CaM (data

PNPP and KH2PO4 both

abolished

1).

the up-regulation

protein

neutral

marked

binding

antagonist

to explain

of CaM on muscarinic

at concentration

due to CaM (Table

postsynaptic

Results

showed

at a concentration

of a CaM mediated protein

effect

CaM antagonists.

on the basal

Similarly,

In order

specific

binding

a suitable

activity

stimulatory

and TFP (10)

reduction

was not

binding

the

we used various

both

(11)

confirm

by CaM we examined

dephosphorylation.

phosphatase

Calcineurin, is known

activity,

of the neuronal

the possibility

membrane

a Ca '+-CaM

to be associated

(12)

may be involved

binding with

in the

of AChR to [3H]-QNB. obtained with

Ni2+,

showed induced

that

preincubation

a dose-dependent 1197

with

calcineurin,

increase

of

previously

[3~1-~~~

the

binding

BIOCHEMICAL

Vol. 133, No. 3. 1985

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

“\

L

/

O/O / P

-I 0

20

60

40

Calcineurin.

pglml

Graph showing the effect of different on the specific binding of [3R]-QNB. Calcineurin (1 r&f) in Tris-buffer for 60 min and the controls of Ni2+-Tris buffer (as 100%). Treatments of the Figure

3.

membrane method.

and

assay

conditions

Mean value

in the phosphorylated a lesser viously

lated thus

synaptic

receptor

activated

with

activation

raised

binding

membrane.

This

the possibility

calcineurin

is

the dephosphorylation several (13).

other Both

brain

concentrations

ized

by inhibitors

may further phosphatase(s)

and high

argue

for

intrinsic

level

the protein

activity to that

(e.g.

synaptic that

such as PNPP,

of inorganic

phosphate

dephosphorylation

to the synaptic

membrane. 1198

3).

To

not preactivators

the calcineurin in prephosphory-

observed

receptor

reported

The fact

(Fig.

to antagonize

substrates

the

(n = 3).

to be potent

by CaM and

up-regulation

may

phosphatase(s)

concentrations

are major the CNS.

1 and

in calcineurin

CaM-dependent

substrates

of phosphatase(s)

mM), ATP (5 I&)

similar

of synaptic

phosphoprotein

within

observed

the CaM induced

CaM and calcineurin

high

is

membrane

Fig.

Tris-buffer

are known

The calcineurin

of its

in

with

phosphatase

effect

that

one.

washed

ATP was found

and its

have been due to the activation which

as described

was also

hand,

amounts of added calcineurin was activated with Ni2' were added equivalent amounts phosphorylated synaptic

was 562 fmol/mg protein

or Mn2+ which

Ni2+

of receptor

synaptic

same

membrane

On the other

of calcineurin.

the

for the Ni2+-control

degree,

activation

are

80

used

in

AChR) are by other

membrane activation

of

this

study

similar

to

investigators

proteins

present

in

by CaM was antagon-

(5 mM); phosphotyrosine (10%)

for

(data

by calcineurin

not

(10 shown)

or related

Vol.

133,

No. 3, 1985

These

BIOCHEMICAL

findings

indicate

and CaM was abolished and Ca2+ within of

[3H]-QNB

to either

binding.

receptor

Calmodulin

Since

by direct

that

CaM down regulates

only

a partial

up-regulation

Burgoyne

(1) by providing the

results

receptor

through

and thus

extend

the original

(1).

Calcineurin

proposed regulation actions

the regulatory

a possible

by protein amongst

(A.K.S.

Heritage Heart

or other

Association,

of AChR.

for Illinois

that reported have

provided

those

up-regulite

AChR.

to demonstrate

although

of

circumstantial, muscarinic

and dephosphorylation,

CaM can only

down regulate

phosphatase(s)

On the basis the

by Burgoyne

complemented

&M-mediating

little

CaM regulates

Our data

study,

states

showed

he was unable

phosphorylation

for

leading

since

that

CaM

of these

cholinergic

may be in-

findings,

muscarinic is

AChR

it

is

receptor

through

the inter-

and the Ca 2+ ion.

work

was supported

Ho) and a Career

Foundation

unlikely

phosphorylation-dephosphorylation

This

CaM (2)

by CaM- Ca2+ ofi cholinergic

observation

mechanism

effect

conditions

in this

of protein

CaM, calcineurin

ACKNOWLEDGEMENTS: award

role

a mechanism

the up-regulation that

appears

CaM can also

presented

of both

in the regulation

a different

using

phosphorylating

that

by phosphorylation

contents

The observations

by phosphatase(s).

strongly

in

it

of CaM regulation

support

activities

protein,

COMMUNICATIONS

on the phosphorylating

chromatography

evidence

induced

to be important

of AChR dependent

AChR under

receptor

loss

to exert

interactions.

explanation

In summary,

appear

the affinity

RESEARCH

The endogenous

appeared

to the immobilized

binding

AChR binding

membrane

down or up regulations

AChR binding

BIOPHYSICAL

by EGTA treatment.

the synaptic

of the membrane.

volved

that

AND

Medical

Scientist

in part award

Research

(J.H.

by a Visiting

Scientist

Wang) from

and a grant-in-aid

from

the Alberta the American

Affiliates.

REFERENCES 1. 2. 3. 4.

Burgoyne, R.D. (1981) FEBS Lett. 127, 144-148. Burgoyne, R.D. (1983) J. Neurochem. 40, 324-331. Stewart, A.A., Ingebritsen, T.S., Manalan, A., Klee, C.B. (1982) FEBS Lett. 137, 80-84. Pallen, C.J. and Wang, J.H. (1985) Arch. Biochem. Biophys. 1199

and Cohen, 237,

P.

281-291.

Vol.

5. 6. 7. 8. 9. 10. 11. 12. 13.

133,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Jones, D.H. and Matus, A.I. (1974) Biochim. Biophys. Acta. 356, 276-287. 102, Sharma, R.K., Taylor, W.A. and Wang, J.H. (1983) Methods in Enzymology 210-221. Pallen, C.J. and Wang, J.H. (1984) J. Biol. Chem. 259, 6134-6141. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Hidaka, H., Sasaki, Y., Tanake, T., Endo, T., Ohno, S., Fujii, Y. and Nagata, T. (1981) Proc. Natl. Acad. Sci. (USA) 78, 4354-4357. Levin, R.M. and Weiss, B. (1977) Mol. Pharmacol. 13, 690-697. Gietzen, K., Adamezyk-Engelmann, P., Wuthrich, A., Konstantinova, A. and Bader, H. (1983) Biochim. Biophys. Acta. 736, 109-118. Wood, J.G., Wallace, R.W., Whittaker, J.N. and Cheung, W.Y. (1980) J. Cell Biol. 84, 66-76. King, M.M., Huang, C.Y., Chock, P.B., Nairn, A.C., Hemmings, Jr. H-C. K.-F Jesse Chan, and Greengard, P. (1984) J. Biol. Chem. 259, 80808083.

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