1

Title: TRANSIENT HYPOXIA INDUCES SEQUESTRATION OF M1 AND M2

mAChRs.

Authors: Liping Mou3, Alicia Gates3, Valerie A. Mosser1, Andrew Tobin2, and Darrell A.

Jackson¶1

1Department of Biomedical and Pharmaceutical Sciences, University of Montana,

Missoula, MT 59812,3Morehouse School of Medicine, Atlanta, Georgia 30310-1495.

2Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box

138, Medical Sciences Building, University Road, Leicester LE1 9HN, United kingdom.

* National Institutes of Health Grants NINDS NS044164, U54-NS 34194, and NCRR

P20 RR15583 supported this work. The costs of publication of this article were defrayed

in part by the payment of page charges.

¶ To whom correspondence and reprint requests should be addressed: Department of

Biomedical and Pharmaceutical Sciences, College of Health Professions and Biomedical

Sciences, Skaggs Building, Room 243, University of Montana, Missoula, MT 59812.

Tel.: 406-243-5761; Fax: 406-243-5228; E-mail address: darrell.jackson@umontana.edu

Abbreviations: CK1α, casein kinase 1 alpha; CHO, Chinese hamster ovary cells; GPCR,

G protein-coupled receptor; GRK2, G protein-coupled receptor kinase 2; mAChR,

muscarinic acetylcholine receptors; NMS, N-methylscoplamine; QNB, quinuclidinyl

benzilate.

Running title: Hypoxia mediated M1 and M2 mAChR sequestration

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ABSTRACT

Oxidative stress has been implicated in impairing muscarinic acetylcholine

receptor (mAChR) signaling activity. It remains unclear whether alteration in the cell

surface distribution of mAChRs following oxidative stress contributes to the diminished

mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in

Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced

oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient

hypoxia was associated with an increase in phosphorylation of these receptors. Over-

expression of a catalytically inactive G-protein-coupled receptor kinase 2 blocked the

increased phosphorylation and sequestration of the M2 mAChR following transient

hypoxia. GRK2 K220R, however, failed to prevent sequestration of the M1 mAChR

following transient hypoxia. Increased phosphorylation and sequestration of the M1

mAChR was blocked by over-expression of a catalytically inactive casein kinase 1 alpha.

These results are the first demonstration that M1 and M2 mAChRs undergo sequestration

following transient hypoxia. The data suggest that increased phosphorylation of M1 and

M2 mAChRs underlies the mechanism responsible for sequestration of these receptors

following transient hypoxia. We report here that distinct pathways involving CK1α and

GRK2 mediate sequestration of M1 and M2 mAChRs following transient hypoxic-

induced oxidative stress.

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INTRODUCTION

Oxidative stress, characterized by exposure to excessive reactive oxygen species, has

been implicated as a key factor in contributing to the neuropathogenesis of varieties of

neurodegenerative diseases and insults that include Alzheimer’s, Parkinson’s, Epilepsy,

and Stroke. Associated with a vast majority of these neurodegenerative injuries is a

hypofunction in the activity of the cholinergic system. Evidence has accumulated

indicating that responses to muscarinic agonists are diminished in primary cortical (Kelly

et al. 1996) (Blanc et al. 1997) (De Sarno and Jope 1998) and immortalized neuronal

cultures (Jope et al. 1999) subject to agents that induce oxidative stress. Collectively,

these studies suggest that impairment of G-protein function due to oxidative stress

underlies diminished cholinergic signaling. This diminished cholinergic signaling appears

to involve dysfunction in G-proteins that mediate phosphoinositide accumulation (Kelly

et al. 1996; Jope et al. 1999). However, it remains unclear whether diminished

cholinergic signaling activities following oxidative stress may also involve alterations in

the levels of cell surface muscarinic receptor numbers. It is known that transferrin

receptors, which internalize through similar endocytotic pathways as mAChRs following

agonist stimulation, undergo redistribution from the cell surface to intracellular

compartments following oxidative stress exposure (Malorni et al. 1998).

Neuroanatomical studies combined with immunohistochemical analysis have

revealed that mAChRs are selectively expressed in the central nervous system. Four of

the mAChR subtypes (M1-M4) have been reported to be present in cholinergic target

fields in the hippocampus (Levey et al. 1991), a region of the brain that is selectively

vulnerable to hypoxic-ischemic induced cell injury and death. The M1 mAChRs shows

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the highest expression of the mAChR subtypes in the hippocampus followed by similar

expression of both the M2 and M4 mAChR subtypes (Levey et al. 1991). To identify

underlying factors that are involved in modulating M1 and M2 mAChRs by transient

hypoxia, we examined the effects of transient hypoxia on Chinese hamster ovarian

(CHO) cells stably expressing the m2 mAChR (Buckley et al. 1989).

We report here that M1 and M2 mAChRs undergo sequestration as a result of

oxidative stress. Additionally, the oxidative stress induced sequestration of the M1 and

M2 mAChRs is associated with an increase in phosphorylation of these receptors. This

study provides new insight into the redistribution of mAChRs in response to oxidative

stress.

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MATERIALS AND METHODS

Materials-[35S]-Methionine (Met; 15.2 µCi/mmol specific activity), [3H]-N-

methylscopolamine (NMS; 81-84 Ci/mmol), [32P]-phosphoric acid (ortho-32P; 200

mCi/mmol) and [3H]-quinuclidinyl benzilate (QNB; 47 Ci/mmol) were purchased from

Amersham Corp. All other chemical used in this study were purchase from Sigma (St.

Louis, MO.).

Cell Culture and Transfection-Chinese hamster ovary (CHO) cells were used throughout

this study. The CHO cell line stably expressing the human M1 and M2 mAChR were

grown as described previously (Buckley et al. 1989). CHO cells were cultured in F10

(Ham) supplemented with 10% fetal bovine serum (FBS) and Penicillin (100

units/ml)/Streptomycin (100 µg/ml). CHO cells were seeded onto 24-well plates at a

density of 8 x 104 cell per well. Approximately 24 h later, cells were transiently co-

transfected with 0.5 µg of enhanced green fluorescent protein (EGFP in pIRES2 was

from Clontech) for transfection efficiency, 0.5 µg Flag-epitope casein kinase 1 α K46R

(F-CK1αK46R in pcDNA-3 was generated by Dr. Andrew Tobin) or 0.5 µg of GRK2

K220R (GRK2 K220R in pcDNA-3 was a gift from Dr. Robert J. Lefkowitz) by

Superfect according to the manufacturer’s protocol (Qiagen). Approximately 70% of the

cells expressed the EGFP.

Hypoxia and re-oxygenation of cultures-Hypoxia was achieved by incubating the

cultures in a controlled atmosphere of 2% oxygen (14 mm Hg partial pressure) for 24 h at

37oC. The single chamber water jacket tissue culture CO2 incubator contained a built in

O2 control system in which O2 levels can be reduced by purging the chamber with pre-

purified nitrogen. The gas mixture in the incubator during hypoxia was 2% O2, 5% CO2,

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and 93% N2. It was determined using an oxygen meter with an O2 microelectrode (OM-4;

Microelectrodes, INC.) that it took approximately 30 min for cultures to become hypoxic

(2% O2) in 24-well plates containing 500 µl of culture media. Re-oxygenation of hypoxic

cultures was accomplished by returning cultures to normoxic (ambient air O2 levels; 21%

O2) tissue culture incubator for various incubation periods as described for individual

experiments.

Saturation binding analysis -The binding of [3H]-QNB to M1 and M2 mAChRs in crude

membrane homogenates was performed as previously described (Halvorsen and

Nathanson 1981) modified from the method of Yamamura (Yamamura et al. 1974). The

assay mixture contained 50 µg of membrane homogenate protein, 10-500 pM [3H]-QNB

in a final volume of 1 ml buffer containing 50 mM NaH2PO4, pH 7.4. Incubation with

[3H]-QNB was carried out at room temperature for 90 minutes. The radioligand-binding

assay was stopped by the addition of 5 ml of ice-cold 50 mM NaH2PO4 to each assay

tube and placing these tubes on ice. Extracts from the tubes were passed thorough a

Whatman glass fiber filter (2.5 cm. GF/C, presoaked in a 0.1% solution of BSA in 50

mM NaH2PO4 buffer). Each assay tube was rinsed 3X with ice-cold 50 mM NaH2PO4

buffer and the GF/C filter was placed in scintillation vials to which 4 ml of scintillation

fluid was added. In all experiments, nonspecific binding was determined as amount of

[3H]-QNB binding remaining in the presence of 1 µM atropine. Protein concentration was

determined by a modification of the procedure of Lowry (Lowry et al. 1951) after

solubilization with sodium deoxycholate and trichloroacetic acid precipitation and using

bovine serum albumin as a standard.

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[3H]-NMS binding assay with intact cells-The binding of [3H]-NMS to intact CHO cell

monolayers was performed as previously described (Nathanson 1983; Feigenbaum and

El-Fakahany 1985). Hypoxic, hypoxic/re-oxygenation and timed-matched normoxic

controls incubations were stopped by aspiration of the media, followed by three washes

with 1-2 ml of ice-cold phosphate-buffered saline (PBS; 137 mM NaCl, 1.68 mM KCl,

1.47 mM KH2PO4, 8.05 mM NaH2PO4; plates were kept on ice from this point on).

Following washes, 1 ml of ice-cold PBS was added to each well (24-well tissue culture

plates). Non-specific binding was determined by the addition of atropine to some of the

wells at a final concentration of 1 µM. Radiolabel [3H]-N-methylscopolamine (NMS)

was added to each well to a final saturation concentration of 0.72 nM. Following

incubation, the assay medium was removed followed by washing each well 3X (1.0

ml/well for a 24-well plate). After the addition of 0.5 ml 1% Triton to each well, cells

were scraped into scintillation vials and 4 ml of scintillation fluid was added to each vial.

Vials were vigorously vortexed and samples were allowed to equilibrate overnight at

room temperature.

Quantitation of cell death-Cell viability was determined using an ethidium homodimer

exclusion test. At the indicated times during hypoxia or hypoxia followed by re-

oxygenation, culture medium was withdrawn and replaced with 300 µl HBBS and

background fluorescence was determined (Fmin). Wells were then brought to 6 µM

ethidium homodimer (Molecular Probes, Eugene, OR) and incubated for 30 minutes at

37ºC at which time fluorescence was measured (F). Finally, wells were brought to 0.03%

saponin and incubated for 60 minutes at 37 ºC and fluorescence was measured a third and

final time (Fmax). Fluorescence was measured with a Spectra Max Gemini XS

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fluorescent plate reader (Molecular Devices, Sunnyvale, CA) at excitation/emission

wavelengths of 530/620 and a cutoff of 610 nm. The percentage of dead cells was

calculated by means of the following formula:

% Dead cells = ((F-F min)/(Fmax – Fmin)) *100. Each measurement was performed in 10

wells and averaged.

Immunoblot analysis of casein kinase 1 α and GRK2 protein expression-Hypoxia and

subsequent re-oxygenation of cultures were terminated by aspiration of the media and

washing cultures twice with ice-cold PBS. The cells were lysed with 50 mM Tris-HCl

buffer (pH 7.4) containing 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM

EDTA, 1 mM PMSF, 1 µg/ml each of Aprotinin, Leupeptin, Pepstatin, 1 mM Na3VO4

and 1 mM NaF. Protein lysates (10 µg) were subjected to 12 % sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to nitrocellulose

membrane (Amersham), blots were incubated with an affinity-purified rabbit polyclonal

antibody for casein kinase 1 α (0.1 µg/ml; provided by Dr. Andrew Tobin; Budd et al.,

2000) or GRK2 (0.1µg/ml; Santa Cruz). As internal controls, blots were probed for β-

actin (Oncogene) using mouse monoclonal antibodies to actin. Immunocomplexes were

visualized by using a peroxidase-conjugated affinity purified goat anti-mouse secondary.

Bands were analyzed using a Kodak imaging software.

[35S]-Methionine metabolic labeling–CHO cells stably expressing the human M2

mAChRs were plated onto 6-well tissue culture plates at a seeding density of 400,000

cells per well. Cultures were rinsed twice and incubated with methionine-free DMEM

medium containing 10% FBS then incubated with 91.2 µCi of [35 S]-methionine (15.2

µCi/mmol specific activity, Amersham) under hypoxia for 24 h or kept under normoxia

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conditions. Following metabolic labeling, cultures were washed 3X with ice-cold PBS

and harvested in 300 µl of 50 mM Tris-HCl buffer (pH 7.4) containing 1% NP-40, 0.25%

Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml each of Aprotinin,

Leupeptin, Pepstatin, 1 mM Na3VO4 and 1 mM NaF buffer. Homogenates were

incubated for 15 minutes at 40C with constant agitation, followed by centrifugation at

14,000 rpm for 15 minutes at 40C. Protein concentrations were determined by the

Bradford method, and lysates (10 µg) were then subjected to electrophoresis on a 10%

SDS-polyacrylamide gel. The gels were vacuumed dry and [35 S]-methionine labeled

protein bands were visualized by autoradiography.

Phosphorylation, immunoprecipitation and autoradiography-CHO M1 and M2 mAChR

cells were seeded onto 6-well tissue culture plates (Falcon) at a seeding density of

800,000 cells per well. Immediately following 24 h of hypoxia, hypoxic and normoxic

control cultures were washed twice with and then incubated 1 h with phosphate-free

DMEM medium. Next, 100 µCi of ortho-32P (200 mCi/mmol specific activity) was added

to each well and incubated for an additional 3 h. Following labeling, cells were washed

3X with ice-cold PBS and harvested in 0.2 ml of buffer A, pH 7.0 (Buffer A: 20 mM

KH2P04, 20 mM NaF, 5 mM EGTA, 5 mM EDTA, 1 mM PMSF, 2.5 µg/ml

Benzamidine, 5.0 µg/ml Leupeptin, 5.0 µg/ml Aprotinin, and 1.0 µg/ml Pepstatin).

Sample homogenates were centrifuged at 14,000 rpm for 10 minutes at 40C and the

supernatant was discarded. The pellets were resuspended in 0.25 ml of buffer B

(Composition of buffer B is the same as buffer A except for the addition of 0.5%

digitonin and 0.05% cholate) and incubated for 60 minutes at 40C with constant agitation.

Following incubation, homogenates were then centrifuged for 10 minutes at 40C.

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Supernatants were removed and transferred to fresh tubes and pre-cleared with 50 µl

protein-A agarose beads (Sigma) for 30 minutes at 40C. Following pre-clearing, samples

were microcentrifuged for 10 minutes at 10,000 rpm at 40C. The supernatant was

removed to a new microcentrifuge tube and the beads were discarded. The supernatants

were aliquot into 3 equal volumes. Each aliquot was immunoprecipitated with M1 or M2

mAChR antibody-agarose conjugate (2.5 µg protein per 25 µl agarose) overnight at 40C

with continuous agitation. The immunoprecipitates were then washed three to five times

with 0.5 ml of buffer C (buffer B containing 200 mM NaCl) and twice with 0.5 ml of

PBS to remove nonspecifically bound proteins. The specifically adsorbed proteins were

eluted from the immunocomplex by incubation in SDS-polyacrylamide gel

electrophoresis sample buffer containing 8 M urea and then subjected to SDS-

polyacrylamide gel electrophoresis on 12% gels containing 4 M urea followed by

electrophoretic transfer to Immobilon-P. Phosphorylation of M1 or M2 mAChR was

visualized by immunoblotting and autoradiography. Analysis of phosphorylated M1 and

M2 mAChR was performed using a Kodak imaging software.

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RESULTS

Transient hypoxia and Ligand Binding-To ascertain whether transient hypoxia causes

sequestration of surface M1 or M2 mAChRs, we performed binding assays in intact CHO

cells that stably expressed the human M1 or M2 mAChR (Buckley et al. 1989) using the

membrane-impermeable muscarinic antagonist [3H]-NMS. After 24 h of hypoxia (2% O2)

incubation, surface M1 and M2 mAChRs were reduced 34% and 36%, respectively,

compared to timed-matched normoxic cultures (Fig. 1A and 1B). Re-oxygenation of

hypoxic cultures to augmented the sequestration of M1 and M2 mAChRs. Both mAChR

subtypes were maximally reduced at 4 h of re-oxygenation. Cell surface M1 and M2

mAChR numbers remained significantly reduced as compared to timed-matched

normoxic cultures for at least 72 h following re-oxygenation (Fig. 1A and 1B). These

data indicate that transient hypoxia causes sequestration of both M1 and M2 mAChRs.

Although saturating concentrations of radioligand were used for all of the binding

experiments, it’s possible that the decreases observed with the radioligand binding

experiments were the result of alterations in mAChR binding affinities. Therefore,

saturation-binding assays were performed to determine whether transient hypoxia alters

the binding affinity of M1 or M2 mAChRs to [3H]-QNB. Transient Hypoxia did not result

in significant alteration in M1 (KD 290.1 pM control vs 334 pM hypoxic) or M2 (KD 60.84

pM control vs 73.4 pM hypoxic) mAChR binding affinity.

Quantitation of cell death-To determines whether hypoxic incubation resulted in

decreased cell viability, we performed an ethidium homodimer exclusion test. Ethidium

homodimer has been used in cytotoxicity assays for several years has very low membrane

permeability unless the integrity of the cell membrane is compromised. As anticipated

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following visual microscopy inspection, 24 h of hypoxic incubation followed by 4 or 24 h

of re-oxygenation had minimal effects on cell viability (see table 1). The percent increase

in dead cells as a result of 24 h of hypoxia incubation followed by either 4 or 24 h of re-

oxygenation was less than 7%. This data provides evidence that sequestration of M1 and

M2 mAChRs by transient hypoxia was not the result of a decrease in cell viability.

Transient hypoxia does not lead to global decrease in protein synthesis- Suppression of

protein synthesis is known to occur in post-ischemic tissues (Krause and Tiffany 1993).

To examine whether global inhibition of protein synthesis occurred as a result of transient

hypoxia, and may underlie the alteration in surface distribution of M1 and M2 mAChRs,

we performed biosynthetic labeling of de novo proteins using [35S]-methionine.

Qualitatively, the majority of [35S]-labeled proteins from hypoxic cultures were not

different from timed-matched normoxic cultures (Fig. 2). This demonstrates that hypoxia

does not lead to global inhibition of protein synthesis in CHO cells. Based upon these

results, sequestration of M1 and M2 mAChRs by transient hypoxia was not due to non-

specific global inhibition of protein synthesis.

Assessment of M1 and M2 mAChR phosphorylation-Previous studies have

demonstrated that agonist-mediated sequestration of the M1 or M2 mAChR was

associated with both receptor subtypes being initially phosphorylated (Tsuga et al. 1994;

Pals-Rylaarsdam et al. 1995; Schlador and Nathanson 1997). Therefore, experiments

were performed to examine whether sequestration of M1 or M2 mAChRs by transient

hypoxia was associated with an increase in phosphorylation of these receptors. Because

both mAChR subtypes were maximally internalized 4 h following re-oxygenation (figure

1A and 1B), phosphorylation experiments were conducted 1 h following re-oxygenation

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and terminated 3 h later (total of 4 h re-oxygenation). Phosphorylation experiments

revealed that transient hypoxia lead to a 45% increase in phosphorylation of the M1

mAChR as compared to M1 mAChRs expressed by normoxic control cells (figure 3A and

3B). Increase in phosphorylation of the M2 mAChRs as a result of transient hypoxia was

even greater than effects seen with M1 mAChRs. Phosphorylation of M2 mAChR was

increased greater than 200% as compared to timed-matched normoxic control (figure 4A

and 4B). This data indicates that sequestration of these mAChR subtypes by transient

hypoxia is associated with an increase in phosphorylation of these receptors.

Assessing the role of GRK2 in mediating M1 and M2 mAChR sequestration by transient

hypoxia-Both receptor subtypes have been reported to be substrates for G-protein-

coupled kinase 2 (GRK2) phosphorylation following agonist stimulation (Haga et al.

1996). For example, transient over-expression of GRK2 facilitates agonist-induced

sequestration of the M1 (Tsuga et al. 1998b) and M2 mAChR (Tsuga et al. 1994; Schlador

and Nathanson 1997; Tsuga et al. 1998a). Additionally, GRK2 activity in rat (Ungerer et

al. 1996) and rabbit hearts has been reported to increase (Maurice et al. 1999) as a result

of oxidative stress induced injury. Therefore, experiments were performed to examine

whether GRK2 had any role in mediating sequestration of M1 or M2 mAChRs by

transient hypoxia. We transiently over-expressed a catalytically inactive GRK2 mutant

(GRK2 K220R), which has been previously shown to act in a dominant negative manner

toward endogenous GRK2 (Tsuga et al. 1994) in CHO cells stably expressing M1 and M2

mAChRs. This GRK2 K220R has been shown previously to attenuate agonist-mediated

sequestration of the M2 receptor (Tsuga et al. 1994). The catalytically inactive GRK2

K220R had no significant effect on the number of M1 or M2 mAChRs expressed on the

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surface of CHO cells as compared to non-transfected control cells (Fig 5A and 5B),

although M1 mAChRs were slightly elevated in GRK 2 K220R transfected cells. GRK2

K220R failed to prevent sequestration of the M1 mAChR by transient hypoxia (Fig 5A),

indicating that GRK2 or GRK2-like kinases were mostly not involved in mediating

sequestration of M1 mAChR by transient hypoxia. In contrast, sequestration of M2

mAChR by transient hypoxia was almost completely blocked in cells transiently

transfected with GRK2 K220R dominant-negative mutant (Fig 5B). These data indicate

that sequestration of M2 mAChRs by transient hypoxia in CHO cells involves GRK2 or a

GRK2-like kinase. In contrast, GRK2 or a GRK2-like kinase is not responsible for

mediating sequestration of the M1 mAChR by transient hypoxia. Collectively, these

results indicate that there are distinct mechanisms mediating sequestration of M1 and M2

mAChRs by transient hypoxia.

Assessing the role of casein kinase 1 α in mediating sequestration of the M1 mAChR

by transient hypoxia-Waugh and Co-workers (Waugh et al. 1999) have reported that in

reconstitution experiments, purified casein kinase 1 alpha (CK1 α) was able to

phosphorylate the M1 mAChR in an agonist-dependent manner in CHO cells.

Additionally, CK1 α was shown to phosphorylate the M3 mAChR in an agonist-

dependent manner (Tobin et al. 1997). To examine whether CK1 α was involved in

mediating sequestration of the M1 mAChRs by transient hypoxia, we over-expressed a

catalytically inactive dominant-negative mutant of CK1 α (CK1 α K46R) in CHO cells

stably expressing the M1 mAChRs. This catalytically inactive mutant of CK1 α has been

shown to inhibit agonist-mediated phosphorylation of the M3 mAChR expressed in either

human embryonic kidney 293 cells (HEK 293) or COS-7 cells (Budd et al. 2000). Over-

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expression of CK1 α K46R blocked sequestration of the M1 mAChRs by transient

hypoxia (figure 6), indicating that CK1 α or a CK1 α-like kinase was involved in the

mechanism underlying sequestration of the M1 mAChR by transient hypoxia.

Affect of transient hypoxia on endogenous CK1 α and GRK2 protein levels-Western

blot analysis was performed to determine whether endogenous CK1 α or GRK2 protein

levels were affected in CHO cells stably expressing the M1 or M2 mAChR, respectively.

There were no alterations in CK1 α protein levels in CHO expressing M1 mAChRs

exposed to 24 h of hypoxia followed by 4 h of re-oxygenation (figure 7A). Similarly,

Western blot analysis of CHO cells stably expressing the M2 mAChRs revealed that 24 h

hypoxia followed by 4 h re-oxygenation did not lead to changes in endogenous GRK2

protein levels (figure 7B). Interestingly, endogenous GRK2 protein levels in CHO cells

stably expressing the M1 mAChRs were nearly absent by 24 h hypoxia followed by 4 h

re-oxygenation incubation (Mou and Jackson 2001). Although GRK2 activity was not

investigated in this study, these data demonstrated that endogenous CK1 α and GRK2

protein levels in CHO cells stably expressing M1 and M2 mAChRs are unaffected by

transient hypoxia. At present, it remains unclear as to the mechanism responsible for

transient hypoxia-induced decrease in GRK2 protein levels in CHO cells stably

expressing the M1 mAChRs.

Assessing whether the inactive CK1 α and GRK2 kinases attenuated the increases in

phosphorylation of M1 and M2 mAChRs by transient hypoxia-Next, we examined

whether the increase in level of phosphorylation of the M1 and M2 mAChRs by transient

hypoxia could be inhibited by exogenously expressing catalytically inactive CK1 α and

GRK2, respectively. Exogenous expression of CK1α K46R did not significantly affect

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the phosphorylation state of M1 mAChR under normoxic conditions (figure 8). Increase,

however, in phosphorylation of M1 mAChR by transient hypoxia was attenuated in

hypoxic cultures expressing the catalytically inactive CK1α K46R (figure 8). Unlike

effects observed with CK1α K46R, expression of GRK2 K220R slightly increased the

phosphorylation state of M2 mAChRs in normoxic cultures (figure 9)., Increase in

phosphorylation of M2 mAChRs by transient hypoxia was blocked by expression of

GRK2 K220R (figure 9). These results indicate that sequestration of M1 or M2 mAChRs

by transient hypoxia involves phosphorylation of these receptor subtypes. Moreover,

these data indicate that phosphorylation and subsequent sequestration of these mAChR

subtypes by transient hypoxia is mediated by CK1 α- and GRK2-like protein kinase,

respectively.

DISCUSSION

Evidence has accumulated indicating that responses to muscarinic agonists are

diminished in primary cortical (Kelly et al. 1996; Blanc et al. 1997; De Sarno and Jope

1998) and immortalized neuronal cultures (Jope et al. 1999) following oxidative stress.

Collectively, these previous studies reported that oxidative stress-induced diminished

mAChR signaling involves impairment of muscarinic receptor activation of G-proteins.

It, however, was not determined whether the decrease in cholinergic signaling by

oxidative stress may also involve alterations in surface mAChR protein levels. Therefore,

experiments were performed to determine whether mAChRs undergo sequestration as a

result of oxidative stress. Experiments were performed on CHO cells that stably

expressed the human M1 or M2 mAChR subtypes. Both mAChR subtypes were

internalized following 24 h of hypoxic incubation. Sequestration of these receptors was

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augmented when hypoxic cultures were re-oxygenated. Saturation radioligand binding

assays indicated that ligand binding affinity was unaffected by transient hypoxia. Kelly

and co-workers (Kelly et al. 1996) also reported that mAChRs ligand binding parameters

are unaffected in cortical neurons when exposed to oxidant such as the amyloid β-

peptide. Interestingly, we also found that total M1 and M2 mAChRs numbers were

unaffected in CHO cells when incubated with hypoxia alone. However, total M1 and M2

mAChRs numbers were reduced in CHO cells following re-oxygenation of hypoxic

cultures. This decrease in total receptor numbers may underlie the augmented decrease in

surface M1 and M2 mAChR protein expression following re-oxygenation. Suppression of

protein synthesis is known to occur in post-ischemic tissue (Krause and Tiffany 1993).

Therefore, the augmented decrease in surface M1 and protein levels may be associated

with a generalized decrease in protein synthesis. However, biosynthetic labeling of

proteins with [35S]-methionine revealed that there was no observable global decrease in

protein synthesis in hypoxic cultures. So, the augmented decrease in surface protein

levels of M1 and M2 may not simply be explained by a generalized global decrease in

protein synthesis. We cannot conclude from these studies that de novo protein synthesis

of M1 and M2 mAChRs wasn’t impaired following transient hypoxia.

G-protein coupled receptor kinase 2 (GRK2) activity has been reported to

increase in membranes of rat (Ungerer et al. 1996) and rabbit hearts (Maurice et al. 1999)

as a result of ischemic injury. In contrast, activity as well as protein levels of GRK2 are

reduced in ischemic canine heart tissue (Yu et al. 2000). Nevertheless, it has been well

established in different cell lines that agonist-induced phosphorylation and subsequent

sequestration of M1 or M2 mAChRs involves GRK2. Experiments were conducted to

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examine whether GRK2 or a GRK2-like protein kinase was involved in mediating

sequestration of M1 and M2 mAChRs by transient hypoxia. Transient over-expression of

the inactive form of GRK2 kinase (GRK2 K220R) blocked sequestration as well as

increased phosphorylation of M2 mAChRs by transient hypoxia. By contrast,

sequestration of M1 mAChRs by transient hypoxia was unaffected by the inactive GRK2.

This demonstrates that distinctly different pathways selectively mediate sequestration of

M1 and M2 mAChRs by transient hypoxia.

Recently, it has been reported in reconstitution experiments that casein kinase 1 α

(CK1α) was able to phosphorylate purified M1 mAChRs in an agonist-dependent manner

(Waugh et al. 1999). Although, there have been no studies to date indicating that CK1 α

activity increases following ischemic injury, CK1 α activity has been reported to increase

as a result of ionizing radiation (Santos et al. 1996). Additionally, CK1α has been

implicated in stabilization of the p53 tumor suppressor protein in response ionizing

radiation, nucleotide depletion, and or hypoxia (Sakaguchi et al. 2000). Therefore,

experiments were performed utilizing a mutant inactive CK1α (CK1α K46R) (Budd et

al. 2000) to examine whether this kinase was involved in mediating phosphorylation and

subsequent sequestration of the M1 mAChR by transient hypoxia. Over-expression of

CK1α K46R attenuated the increase in phosphorylation as well as blocked the

sequestration of the M1 mAChR by transient hypoxia.

Both mAChR subtypes have been shown to undergo heterologous receptor

regulation. For example, Habecker and Nathanson (Habecker and Nathanson 1992)

demonstrated in embryonic chick cardiomyocytes that surface M2 mAChRs are reduced

following agonist-mediated activation of adenosine A1 receptors. Therefore, transient

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hypoxia-induced phosphorylation and sequestration of M1 and M2 mAChRs may involve

release of substances such as adenosine. Chronic hypoxia has been shown to result in an

increased release of adenosine in human fibroblast (Reisert et al. 2002) and rat PC12 cells

(Kobayashi et al. 2000). Additionally, adenosine, which has been reported to accumulate

in the synapse of hippocampal CA1 neurons during hypoxia (Doolette 1997) (Pearson et

al. 2001), has been implicated with impairing interneuronal M2 mAChRs function.

However, stimulation of CHO cells with the adenosine analog 5’-N-

ethylcarboxamidoadenosine (NECA), known to act at A1, A2A, A2B, and A3 adenosine

receptor subtypes was reported not to have any effect on untransfected CHO cells, but did

result in a time- and dose-dependent phosphorylation of extracellular-regulated kinase 1/2

(ERK1/2) in CHO cells transfected with each adenosine receptor subtype (Schulte and

Fredholm 2000). Because of the lack of endogenous adenosine receptor subtypes,

adenosine does not appear as a likely candidate in mediating sequestration of M1 or M2

mAChRs by transient hypoxia in CHO cells. Studies are ongoing to determine whether

increased phosphorylation and sequestration of M1 and M2 mAChRs by transient hypoxia

involves release of as yet unidentified substance(s).

In summary, we have demonstrated that M1 and M2 mAChRs undergo

internalization by transient hypoxia. Furthermore, phosphorylation appears to mediate

sequestration of these receptors by transient hypoxia. Lastly, distinct and selective

pathways mediate phosphorylation and subsequent sequestration of the M1 and M2

mAChRs. CK1α or CK1α-like kinase is involved in mediating sequestration of the M1

mAChR. While, GRK2 or GRK2-like kinase is involved in mediating sequestration of the

M2 mAChR by transient hypoxia.

20

21

ACKNOWLEDGEMENT

We thank Dr. R.J. Lefkowitz for cDNA of mutant GRK2 (GRK2 K220R), Dr.

Tom Bonner for the CHO cells stably expressing the human M1 and M2 mAChR, and Dr.

P. MacLeish and Dr. J. Blusztajn for comments and editing the manuscript.

22

REFERENCES

FIGURES AND LEGENDS

Figure 1

A

B

23

Figure 1. Transient hypoxia causes sequestration of surface M1 and M2 mAChRs.

CHO cells stably expressing human M1 (A) or M2 (B) were subjected to hypoxia (2% O2)

for 24 h followed by different re-oxygenation time periods. [3H]-NMS radioligand

binding to intact cells was performed as described under “Method Section”. Data are

presented as mean ± standard deviation from two to three separate experiments with each

experiment consisting of 8-11 determinants. There were significant differences between

normoxic (closed square) and re-oxygenated cultures (open square) at all normoxic time

points (p<0.001; ANOVA with post hoc Bonferroni/Dunn test).

24

% Cell Death H/R M1 M2

24/4 6.08 ± 1.03

4.18 ± 1.46

24/24 1.66 ± 0.89 2.93 ± 1.10

Table 1. Transient hypoxia does not significantly affect cell viability. CHO cells

stably expressing human M1 or M2 were subjected to hypoxia (2% O2) for 24 h followed

by re-oxygenation for 4 (24/4) or 24 (24/24) hours. Cell viability was determined using

an ethidium homodimer assay as described under “Method Section”. Data are presented

as mean ± standard error from two separate experiments with each experiment consisting

10 determinations. (H/R; hypoxia/re-oxygenation)

25

Figure 2

Lane # 1 2 3 4

Figure 2. Effects of hypoxia on protein synthesis. [35 S]-methionine metabolic labeling

of cells kept under hypoxic or normoxic conditions as described under “Method Section”.

Lanes consisted of 24 h hypoxic (lanes 1 and 3) and time-matched normoxic cultures

(lanes 2 and 4).

26

Figure 3

B

Lane # 1 2 3 4 5 6 AA

*

27

Figure 3. Increase phosphorylation of CHO M1 mAChRs by transient hypoxia.

Cultures were labeled with 100 µCi/well ortho 32P 1 h following re-oxygenation for 3 h.

Phosphorylation of M1 mAChRs is revealed by autoradiography after

immunoprecipitation and SDS-PAGE gel electrophoresis. A.) Lanes consisted of timed-

matched normoxic control cultures (lanes 1 and 2), 1mM carbachol stimulation for 60

min (lanes 3 and 4), and 24 h hypoxia followed by 4 h of re-oxygenation (Lanes 5 and 6).

The autoradiogram is a representative of similar autoradiograms from three independent

experiments. B.) Quantitation of transient hypoxia mediated increase in phosphorylation

of M1 mAChRs is shown in histogram in which image analysis of band intensities were

performed from three independent experiments that were performed in duplicates.

Phosphorylation of M1 mAChRs was significantly increased in transient hypoxic cultures

(25697 ± 3417, band intensity, arbitrary units) by 45% over normoxic cultures (17642 ±

2308; Asterisk indicates statistical significance; p<0.006, Paired t-test). Data are

presented as the mean ± standard deviation from three separate experiments.

28

Figure 4

A

B

Lane # 1 2 3 4 5 6

*

29

Figure 4. Increase in phosphorylation of CHO M2 mAChRs by transient hypoxia

Cultures were labeled with ortho-P32 as described in figure 3 and method section. A.)

Lanes consisted of transient hypoxic cultures (lanes 1, 3, and 5) and timed-matched

normoxic control cultures (lanes 2, 4, and 6). The autoradiogram is a representative of

similar autoradiograms from three independent experiments. B.) Quantitation of transient

hypoxia mediated increase in phosphorylation of M2 mAChRs is shown in histogram in

which image analysis of band intensities were performed from three independent

experiments that were performed in duplicates. Phosphorylation of M2 mAChRs was

significantly increased in transient hypoxic cultures (8253 ± 182.35, band intensity,

arbitrary units) nearly 2-fold over normoxic cultures (4752.64 ± 1067.7; Asterisk

indicates statistical significance; p<0.006, Paired t-test). Data are presented as the mean ±

standard deviation from three separate experiments.

30

Figure 5

A.

B.

NS

* NS

* NS

31

Figure 5. Over-expression of dominant-negative GRK2 blunts transient hypoxia

mediated sequestration of M2 mAChRs but not M1 mAChR. Cells were co –

transfected with 0.5 µg of pcDNA3-GRK2 K220R (GRK2DN) and 0.5 µg of pIRES2-

EGFP for transfection efficiency as described in the method section. Twenty-four hours

following transfection, CHO cells stably expressing the M1 (A) or M2 mAChRs (B) were

placed under hypoxia for 24 h followed by 4 h of re-oxygenation. Surface mAChRs were

measured with the impermeable muscarinic antagonist [3H]-N-methylscopolamine

([3H]-NMS). Data are presented as the mean ± standard deviation from three separate

experiments with each experiment consisting of 8-11 determinants. Statistical

comparisons indicated significance between normoxic, hypoxic/re-oxygenated groups

(p<0.0001; ANOVA with post hoc Bonferroni/Dunn test) and hypoxic/re-oxygenated,

hypoxic/ re-oxygenated-transfected groups (Asterisks *, **, indicate a p< 0.0001;

ANOVA with post hoc Bonferroni/Dunn test; NS, not significant).

32

Figure 6

Figure 6. Transient hypoxia mediated sequestration of CHO M1 mAChRs is blocked

by dominant negative CK1 α. Following 24 h of transfection; CHO cells stably

expressing the M1 mAChR were placed under hypoxia for 24 h followed by 4 h of re-

oxygenation. Data are presented as mean ± standard deviation from three separate

experiments that consisted of 8-10 determinants. Statistical comparisons indicated

significance between normoxic, hypoxic/re-oxygenated groups (p<0.0001; ANOVA with

post hoc Bonferroni/Dunn test). There were no significant differences between normoxic

transfected and hypoxic/ re-oxygenated-transfected groups (Asterisk * indicate a p<

0.0001; ANOVA with post hoc Bonferroni/Dunn test; NS , not significant).

33

Figure 7

A.

B.

34

Figure 7. Transient hypoxia does not alter endogenous CK1 α or GRK2 protein

levels. A representative Western blot from three independent experiments reveals that

there were no observable changes in CK1 α (A) or GRK2 (B) protein levels between

normoxic and hypoxic/ re-oxygenated cultures. Lanes consisted of timed-matched-

normoxic controls (Lanes 1, 3, and 5) and twenty-four hours of hypoxia followed by 4

hours of re-oxygenation (Lanes 2, 4, and 6).

35

Figure 8

Figure 8. Expression of a dominant negative form of CK1α blocks increase in M1

phosphorylation by transient hypoxia. Autoradiogram shown here is representative of

at least three individual experiments. Following 24 h of transfection with dominant

negative inactive casein kinase 1 alpha, cultures were placed or not under hypoxia for 24

h. Ortho-32P was added 1 h into re-oxygenation and experiment was terminated following

4 h of re-oxygenation. Lanes consisted of 30 min 10-4 carbachol (lane 1 and 2), timed

match normoxia (lane 3 and 4), timed match normoxia transfected with dominant

negative CK1 α (lane 5 and 6), 24 h hypoxia, 4 h re-oxygenation (lane 7 and 8), and 24 h

hypoxia, 4 h re-oxygenation transfected with CK1 α (lane 9 and 10).

36

Figure 9

Figure 9. Over-expression of dominant-negative form of GRK2 blunts increase of

phosphorylation of M2 mAChR by transient hypoxia. Lanes consisted of transient

hypoxic cultures (24 h hypoxia and 4 h normoxic incubation) not transfected (lanes 1 and

3), timed-matched normoxic cultures not transfected (lanes 2 and 4), transfected

normoxic cultures (lanes 5 and 7), transfected transient hypoxic cultures (lanes 6 and 8),

and 30-min 1mM carbachol stimulated cultures (positive control, lanes 9-10). The white

arrow indicates phosphorylated M2 mAChR bands. The autoradiogram is a representative

of three independently conducted experiments.

37

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