J. OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH, 20( l) , 75-85 (2000)

FUNCTIONAL RECEPTOR COUPLING TO G, IS A MECHANISM OF

AGONIST-PROMOTED DESENSITIZATION OF THE

P2-ADRENERGIC RECEPTOR

Nicole M. Tepe and Stephen B. Liggett. Departments of Pharmacology and Medicine University of Cincinnati College of Medicine

23 1 Bethesda Avenue Cincinnati, OH 45267-0564

ABSTRACT

The Pz-adrenergic receptor (p2AR) couples to Gs, activating adenylyl cyclase (AC) and increasing CAMP. Such signaling undergoes desensitization with continued agonist exposure. P2AR also couple to Gi after receptor phosphorylation by the CAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of P2AR-Gi coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express P2AR or P2AR and Gia2, and then treated with vehicle or the agonist isoproterenol to evoke agonist- promoted p2AR desensitization. Membrane AC activities showed that Gia2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist- stimulated AC was observed with the cells overexpressing Gid. In the absence of such overexpression, P2AR desensitization was 23f7%, while with 5-fold G,d overexpression desensitization was 58+5% (p<O.Ol, n 4 ) . The effect of Gi on desensitization was receptor-specific, in that forskolin responses were not altered by Gia2 overexpression. Thus, acquired PzAR coupling to Gi is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase G, levels contribute to p2AR dysfunction.

75

Copyright 0 2000 by Marcel Dekker. Inc. www .dekker.com

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76 TEPE AND LIGGETT

INTRODUCTION

Desensitization is defined as the waning of a signal despite continued

presence of a stimulus. Like many G-protein coupled receptors, the P2-adrenergic

receptor ( P A R ) displays desensitization during continuous exposure to agonists

(1). The process is initiated within seconds to minutes of agonist exposure. This

rapid form of desensitization has been shown, in part, to be due to

phosphorylation of the receptor which acts to uncouple the receptor from its

cognate G-protein, G, (2). This results, then, in a decrease in agonist-stimulated

adenylyl cyclase activity and intracellular CAMP levels. Agonist-promoted

phosphorylation of the P 2 A R occurs via the PAR kinase (PARK) and protein

kinase A (PKA) (2). The latter represents a classic negative feedback loop, in that

the second messenger that is increased as a consequence of signal transduction

serves as the trigger for signal attenuation. In contrast, PARK mediated

phosphorylation of P z A R is not dependent on CAMP, but rather requires that the

receptor be in the active (agonist-occupied) conformation.

Several studies have suggested that P2AR coupling to the inhibitory G-protein,

Gi, may also be a mechanism of desensitization although the hypothesis has not

been explicitly tested. In peptide based studies, it has been shown that following

PKA phosphorylation a peptide identical to a portion of the third intracellular loop

of the P2AR binds G,i (3). Furthermore, P z A R expressed in HEK293 cells couple

to activation of the mitogen activated protein kinase (MAP kinase) pathway,

presumably via the Py derived from Gi (4). Interestingly, in animal models of

heart failure and asthma, two diseases where P z A R dysfunction is known to occur,

G, has been found to be elevated in heart (5,6) or lung (7,8). This has given rise

to the idea that P z A R coupling to Gi may decrease basal or agonist-mediated

coupling of the receptor to the stimulation of adenylyl cyclase, or may be a

component of desensitization. To directly test the notion that PZAR-Gi coupling

occurs and is a mechanism of agonist-promoted desensitization, we canied out

studies using HEK293 cells transfected to overexpress P z A R , or PzAR along with the a subunit of Gi2.

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DESENSITIZATION OF P,-ADRENERGIC RECEPTOR 77

MATERIALS AND METHODS

Materials: HEK293 cells were from American Type Culture Collection.

Pertussis toxin was from Sigma. Tissue culture media and antibiotics were from

Gibco. '251-CYP, [3H]cAMP, and [a3P]ATP were from New England Nuclear.

Transfections and cell culture: HEK293 cells were transfected using a calcium

phosphate technique as previously described (9). Constructs consisted of the

human PzAR in the expression vector pBCl2BI (1 0) and the rat Gja2 in the vector

pCDNA1. Cells were transfected with 10 pg of the P2AR construct alone or 10

pg of the p 2 A R construct and 5 pg of the Ga,z construct and studied two days

later. Cells were maintained in Dulbecco's modified Eagles medium with 10%

fetal calf serum in a 95% air, 5% C02 atmosphere at 37°C .

Immunoblots: Polyclonal antisera against Gai2 (Santa CW) was used at a titer of

1 :200 in immunoblots to detect the level of expressed Gaj2 in whole cell lysates as

described (1 1). Blots were developed by enhanced chemiluminescence and

quantitated using Scan Analysis software.

Radiolinand binding: Cells in monolayers were washed three times with

phosphate buffered saline and detached and disrupted by scraping with a rubber

policeman in 5 m M Tris, pH 7.4, 2mM EDTA buffer at 4". The suspension was

centrifiged once at 38,000 xg for 10 minutes and the membranes resuspended in

75 mM Tris, pH 7.40, 5mM MgC12, 2mM EDTA buffer at 25". Receptor density

was determined by radioligand binding with 251-cyanopindolol ( ' 2 5 1 - C ~ ) as

described (12) at 25" for two hours. Bound '251-CYP was separated from free by

rapid vacuum filtration over glass fiber filters.

Adenvlvl Cvclase Activity: Membranes prepared as above were resuspended in a

buffer that provided for 30 mM Tris, pH 7.4, 2 mM MgCl2 and 0.8 mM EDTA in

the final reaction. Activities were determined in a reaction which consisted of

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78 TEPE AND LIGGETT

membranes (-10 pg), 120 uM ATP, 60 uM GTP, 2.8 mM phosphoenolpyruvate,

50 pg/ml myokinase, 100 pM CAMP and 2 pCi [d2P]ATP for 30 minutes at 37"

as described (13). Reactions were carried out with various concentrations of

isoproterenol, water (basal), or 100 pM forskolin. [32P]cAMP was separated from

[32P]ATP by column chromatography over alumina columns. A [3H]cAMP

standard included in the stop buffer was used to account for individual column

recovery. Protein determination was by the copper bicinchoninic acid method

(14).

STATISTICAL ANALYSIS

Dose-response curves were analyzed by an iterative least-squares technique

using Prizm software (San Diego, CA). Statistical comparisons were made by

paired or unpaired t-tests as appropriate. Significance was imparted when p<0.05.

Data are presented as mean f standard error (SE).

RESULTS

Our initial efforts utilized pertussis toxin, an agent that ADP-ribosylates Gai

rendering it unable to couple to receptors (15). The approach was to expose cells

in culture overnight to 500 ng/ml of the toxin and then assess the effects on p 2 A R

coupling to adenylyl cyclase. As shown in figure 1, basal and isoproterenol (as

well as forskolin) stimulated adenylyl cyclase activities were increased with

pertussis toxin treatment. Although absolute levels of adenylyl cyclase activity

were increased, the fold-stimulation by isoproterenol over basal was not affected

by toxin (2.27f0.04 fold in the absence and 2.401t0.17 fold in the presence of the

toxin pN.05). This sensitization of adenylyl cyclase by pertussis toxin, which

has been previously described (16,17), makes it difficult to assess receptor

coupling under these conditions. Agonist-promoted desensitization would be

particularly problematic, since the enhanced adenylyl cyclase activity elevates

CAMP and thus PKA activity, which would serve to desensitize the p 2 A R prior to

agonist exposure.

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DESENSITIZATION OF Pz-ADRENERGIC RECEPTOR 19

koproterenol, Log M

FIG 1. Pertussis toxin sensitizes adenylyl cyclase but does not increase P z A R stimulation of adenylyl cyclase over basal activities. HEK293 cells were incubated with 500 ng/ml pertussis toxin or vehicle for 18 hours at 37", membranes prepared, and adenylyl cyclase activities determined. The fold stimulation by isoproterenol over basal levels was not increased by pertussis toxin. Shown are results of 4 experiments.

Given the above, we next turned to a strategy whereby P2AR was expressed

alone, or P z A R and Gaiz were co-expressed, in HEK293 cells. P2AR was

overexpressed by >50 fold, so that the ratio of receptor to Gai2 would be increased

and the G protein became a limiting factor. With concomitant overexpression of

Gaiz, then, an augmentation of any effects of receptor-Gai coupling would be

expected if such coupling indeed occurred. The amount of the Gai2 construct used

for the transfections was adjusted such that Gai2 was overexpressed -5 fold over

background (figure 2).

The effects of such overexpression of Gail on adenylyl cyclase activity are

shown in figure 3. Basal levels of activity were 9.06f1.04 pmol/min/mg in the

absence of Gal2 overexpression and 6.91fl. 1 pmol/min/mg with overexpression

( ~ ~ 0 . 0 5 , n= 4). Maximal agonist-stimulated activities were also lower, but the

fold-stimulation of adenylyl cyclase over basal by agonist (2.23f0.12 fold) was

not changed by concomitant Gai2 overexpression (2.28f0.16 fold, pN.05).

Given that it is the PKA phosphorylated form of the P z A R that is proposed to

actively couple to Gi, we next examined the effect of Gai2 overexpression on

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80 TEPE AND LIGGETT

Gia, --+

FIG 2. Overexpression of Gla2 in HEK293 cells. Cells were transfected with vectors to express p2AR alone, or p2AR and Gla2. Two days later whole cell lysates were prepared and fractionated on 10% SDS-polyacrylamide gels, transferred to membranes, and immunoblotted for quantitation of Glaz. The p2AR 3 Gla2 cells overexpressed Gla2 -5 fold over Pp4.R cells and nontransfected cells.

5 - I

0

1 -3

PzAR P2AR + Gi

isoproterenol, Log M

FIG 3. Effects of G, overexpression on p2AR coupling to the stimulation of adenylyl cyclase. Membranes were prepared from transfected cells expressing p2AR alone or p2AR and Gia2 and adenylyl cyclase activities determined. The fold-stimulation by isoproterenol over basal levels was not altered by Gla2 overexpression. Shown are results from four experiments.

p2AR signalling after treatment of the cells with 10 pM isoproterenol for 30

minutes in culture. Cells were then washed with ice-cold saline, membranes

prepared, and adenylyl cyclase activities determined. In the absence of GaI2

overexpression, this agonist treatment resulted in a 2327% desensitization of the

maximal isoproterenol response (figure 4a). In marked contrast, when Gal2 was

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DESENSITIZATION OF P2-ADRENERGIC RECEPTOR 81

A.

~++/'.;o :Q -i 17 i .5 i Iroproterenol, Log M

B. P2AR +Gi

; 20j 0 Control

0 1 ' 1 , I , I I I I I 0 -10 -0 6 -7 4 4 -4 -3

Isoproterand, Log M

FIG 4. GI overexpression enhances agonist promoted desensitization of the p2AR. Cells expressing p2AR alone or p 2 A R and Gla2 were treated with vehicle or the agonist isoproterenol (10pM) for 30 minutes at 37", membranes prepared, and adenylyl cyclase activities determined. Overexpression of Gia2 enhanced desensitization from 23+7% to 58+5%. Shown are the results of four experiments. *p<O.O 1 compared to cells overexpressing only p 2 A R .

overexpressed -5 fold, maximal agonist stimulated adenylyl cyclase activity was

found to be significantly reduced after agonist treatment (figure 4b). This

amounted to a 58*5% desensitization (p<O.Ol vs desensitization without Gia2).

Thus after agonist exposure, the p 2 A R attained the ability to functionally couple

to G, and inhibit adenylyl cyclase, evoking enhanced desensitization. Indeed, the

extent of desensitization due to overexpressed Gai2 was more than double that

observed with native GI expression. On the other hand, non-receptor stimulated

adenylyl cyclase activities by forskolin were unaffected by agonist treatment in

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82 TEPE AND LIGGE'IT

cells only overexpressing p2AR (control = 75.3rt7.5 pmol/min/mg, agonist treated

= 77.5f11.1 pmol/min/mg) and this pattern was not altered by concomitant

overexpression of Gai2 (60.4f7.5 vs 57.3rt7.5 pmol/min/mg, respectively).

DISCUSSION

Agonist-promoted desensitization of G-protein coupled receptors such as the

P2-adrenergic receptor (p2AR) serves to assimilate the many signals received by

the cell in order to maintain homeostasis, but may also be maladaptive in certain

diseases or in tachphylaxis to therapeutic agonists (1,lS). A recent report

demonstrates that p2AR, which are classically considered to be coupled to G, and

the stimulation of adenylyl cyclase, are also coupled to the stimulation MAP

kinase (4). This signalling was found to be acquired by the receptor via

phosphorylation by PKA, which results in coupling of p2AR to the inhibitory G- protein, Gi. While such multifunctional signalling has implications relative to this

new pathway, we have considered that agonist-dependent Gi coupling may serve

another role. Since G, and Gi have opposing effects on the activity of adenylyl

cyclase, this switch to Gi coupling also has the potential to participate in a

dampening of the stimulatory signal to adenylyl cyclase and thus could represent

an additional mechanism of agonist-promoted desensitization of the p 2 A R .

Furthermore, if such P2AR.-Gi coupling indeed has this function, increases in the

cellular levels of Gi in certain diseases might be a mechanism of enhanced p2AR

desensitization. Indeed, in animal models of heart failure (19,20) and the human

syndrome (5,6), Gi levels have been observed to be increased on the order of

several fold. Whether such increases are a specific mechanism of p2AR

desensitization, though, are not known. Similarly, in animal models of asthma (7,

8), p2AR mediated signaling is depressed and Gi levels of the large airways are

increased due to passive sensitization and cytokine exposure. Clearly in both of

the above diseases, desensitization of the p2AR in cardiomyocytes or bronchial

smooth muscle, respectively, occurs and acts to limit receptor function. In heart

failure, this desensitization limits cardiac inotropy and chronotropy and the

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DESENSITIZATION OF P,-ADRENERGIC RECEPTOR 83

response to endogenous and exogenously administered agonists. Such

desensitization may be maladaptive at certain stages of the disease, but may also

serve a protective function in advanced disease by minimizing cardiac energy

expenditure in the face of limited metabolic reserves (21). In asthma, P 2 A R

desensitization by increased Gi may occur through receptor crosstalk or immune

stimulated mechanisms, and leads to increased bronchial obstruction due to

depressed function of the major bronchodilating receptor of the airway. In

addition, the response to P-agonists, the most commonly utilized agents used for

the treatment of obstructive airways diseases, is attenuated under these conditions.

In the current study we show that an increase of Gj expression of -5 fold,

which is similar in magnitude to that observed in heart failure and asthma, causes

enhanced agonist-promoted desensitization of p 2 A R function. The increase in Gi

was found to lower adenylyl cyclase activities determined in cell membranes.

However, the fold-stimulation by isoproterenol over basal was not altered by

increased G, expression. We thus conclude that in the absence of conditions

where PKA is activated, that P2AR-Gi coupling is insignificant. However when

PKA is activated in the cell, such as with persistent agonist exposure or with

activation of other G, coupled receptors, P2AR-Gi coupling is of sufficient

efficiency to decrease adenylyl cyclase activity. As such, this affects the increase

promoted by PZAR-G, coupling and thus P2AR-Gi coupling can be a significant

component of desensitization of P2AR signaling to the stimulation of adenylyl

cyclase.

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