[methods in enzymology] constitutive activity in receptors and other proteins, part b volume 485 ||...

C H A P T E R S E V E N

M

IS

*{

ethods

SN 0

DepaDepa

Constitutive Activity and Inverse

Agonism at the a1a and a1b AdrenergicReceptor Subtypes

Susanna Cotecchia*,†

Contents

1. In

in

076

rtmrtm

troduction

Enzymology, Volume 485 # 2010

-6879, DOI: 10.1016/S0076-6879(10)85007-4 All rig

ent of General and Environmental Physiology, University of Bari, Italyent of Pharmacology and Toxicology, University of Lausanne, Switzerland

Else

hts

124

2. C
ombination of Computational Modeling and Site-Directed
Mutagenesis of the Receptor to Identify Constitutively

Activating Mutations
127
2
.1. T he predictive ability of molecular modeling: The E/DRY motif 127
2
.2. M icrodomains involved in receptor activation 128
2
.3. C onstitutively activating mutations of the a1a- anda1b-AR subtypes 129
3. M
easuring Constitutive Activity of Receptor-Mediated Gq Activation 130
3
.1. C onstitutive activity of a1-AR CAMs 130
3
.2. C onstitutive activity of wild-type a1-ARs 132
4. In
verse Agonism at the a1-ARs 132
5. C
onclusions 135
Ackn
owledgments 136
Refe
rences 136
Abstract

The a1b-adrenergic receptor (AR) was, after rhodopsin, the first G protein-

coupled receptor (GPCR) in which point mutations were shown to trigger

constitutive (agonist-independent) activity. Constitutively activating mutations

have been found in other AR subtypes as well as in several GPCRs. This chapter

briefly summarizes the main findings on constitutively active mutants of the

a1a- and a1b-AR subtypes and the methods used to predict activating mutations,

to measure constitutive activity of Gq-coupled receptors and to investigate

inverse agonism. In addition, it highlights the implications of studies on consti-

tutively active AR mutants on elucidating the molecular mechanisms of receptor

activation and drug action.

vier Inc.

reserved.

123

124 Susanna Cotecchia

Abbreviations

AR
adrenergic receptor CAM constitutively active mutant GPCR G protein-coupled receptor IP inositol phosphate MD molecular dynamics
1. Introduction

Within the large family of G protein-coupled receptors (GPCRs), theadrenergic receptors (ARs) include nine gene products: three b (b1, b2, b3),three a2 (a2A, a2B, a2C), and three a1 (a1a, a1b, a1d) receptor subtypes. Thea1-AR subtypes (Schwinn et al., 1995) mediate several important centraland peripheral effects of epinephrine and norepinephrine including thecontrol of blood pressure, heart growth, glucose metabolism, and variousbehavioral responses.

Mutational analysis performed by various investigators contributed toidentify some of the structural determinants of the a1-AR subtypes involvedin each of the three main “classical” functional properties of GPCRs:(1) ligand binding (Cavalli et al., 1996; Hwa and Perez, 1996); (2) couplingto G protein-effector systems (Cotecchia et al., 1992; Greasley et al., 2001);(3) desensitization (Diviani et al., 1996, 1997; Lattion et al., 1994; Vazquez-Prado et al., 2000).

Activation of the a1-AR subtypes causes polyphosphoinositide hydro-lysis catalyzed by phospholipase C via pertussis toxin-insensitive G proteinsof the Gq/11 family in almost all tissues where this effect has been examined.Polyphosphoinositide hydrolysis results in the increase of intracellular inosi-tol phosphate (IP) production. Several lines of evidence demonstrated thatthe third intracellular (i3) loop contains the main structural determinantsinvolved in a1b-AR coupling to G proteins of the Gq/11 family. In particular,the results from site-directed mutagenesis in conjunction with the predic-tions of molecular modeling suggested that specific residues, that is, R254 andK258 in the i3 loop as well as L151 in the second intracellular loop, are directlyinvolved in receptor-G protein interaction and/or receptor-mediatedactivation of the G protein (Fanelli et al., 1999; Greasley et al., 2001). Specificresidues involved in receptor-G protein coupling have not been identifiedyet in the a1a- and a1d-AR subtypes.

In the context of a study in which a number of residues in the i3 loop of thea1b-AR were mutated, it was fortuitously discovered that a conservative

Constitutive Activity at a1-Adrenergic Receptors 125

substitution (A293L) in the cytosolic extension of helix 6 (Fig. 7.1) resulted inagonist-independent receptor activation of polyphosphoinositide hydrolysis(Cotecchia et al., 1990). The a1b-AR was, after rhodopsin, the first GPCRin which point mutations were shown to trigger constitutive (agonist-independent) receptor activation. To further assess the role of A293, thisamino acid was systematically mutated by substituting each of the other 19amino acids (Kjelsberg et al., 1992). Remarkably, all possible amino acidsubstitutions of A293(6.34) (the superscript refers to numbering fromBallesteros and Weinstein, 1995) in the a1b-AR induced variable levels ofconstitutive activity which was the highest for the A293E mutant (Fig. 7.2).

Similar mutations were performed in the b2 (Samama et al., 1993) anda2AAR (Ren et al., 1993) leading to increased or decreased agonist-independent adenylyl cyclase activity, respectively.

NE269(6.30)

(“ionic lock” with R143)

E C

C

1 32 4 5 6 7

RY

D A

K R

254258

A293

D142

R143

E289

A293

F303

Helix 3Helix 6

Intracellular

Extracellular

a1b-adrenergic receptor

D142(3.49)

NPXXY(X)5,6F

Figure 7.1 The a1b-adrenergic receptor (AR). (Left) Topographical model of thereceptor displaying key amino acids involved in receptor function mentioned in thetext. (Right) Relative position of helices 3 and 6 in the homology model of the wild-type a1b-AR. Comparative modeling and molecular dynamic simulations were per-formed as described in Greasley et al. (2002). The view displays the amino acids ofhelices 3 and 6 involved in receptor activation. Van der Waals spheres, whose radiushas been reduced by 40%, depict each side chain. The color indicates the effect ofmutations: white/no effect, green/constitutively activating, red/impairing receptorsignaling, violet/either impairing or constitutively activating depending upon thesubstituent amino acid.

0

5000

10,000

15,000 BASAL

EPI

WT D142A

[3H

]-IP

acc

umul

atio

n (c

pm)

A293E E289A

Figure 7.2 Constitutively active mutants of the a1b-AR. COS-7 cells (0.3 � 106)were seeded in 6-well plates and transfected with different amounts of DNA encodingthe receptors (0.2 mg/million cells for wild type, A293E, and E289A, 2 mg/million cellsfor D142A) to obtain comparable levels of expression. The receptor densities measuredon membrane preparations were �200 fmol/million of cells. Cells were harvested 48 hafter transfection. Transfected cells were labeled for 15–18 h with myo-[3H]inositol at5 mCi/ml in inositol-free DMEM supplemented with 1% fetal bovine serum. Cells werepreincubated for 10 min in PBS containing 20 mM LiCl and then treated with differentligands. Total inositol phosphates (IPs) were extracted and separated as previouslydescribed (Cotecchia et al., 1992). IP accumulation was measured in cells expressingthe wild-type a1b-AR and its mutants D142A, A293E, and E289A in the absence(BASAL) or in the presence of 10�4

M epinephrine (EPI) for 45 min.


The discovery of constitutively active mutants (CAMs) in the AR familyhad several implications (Costa and Cotecchia, 2005). First, it highlightedconstitutive activity as a potential intrinsic feature of GPCRs. Second, itencouraged the search for spontaneous activating mutations involved in thepathogenesis of diseases. Third, it lead to the identification of inverseagonism, that is, the capacity of ligands, previously characterized as ARantagonists, to inhibit the constitutive activity of the receptors.

In this chapter, we will review some of the findings obtained in the pastseveral years on constitutive activity and inverse agonism at the a1a- anda1b-AR subtypes. A different chapter of this volume is devoted to the a1d-AR subtype. We will focus our attention on three methodological issues:(i) combination of computational modeling and site-directed mutagenesisof the receptor to identify constitutively activating mutations; (ii) measuringconstitutive activity of receptor-mediated Gq activation; (iii) measuringinverse agonism.


2. Combination of Computational Modeling and

Site-Directed Mutagenesis of the Receptor to

Identify Constitutively Activating Mutations

2.1. The predictive ability of molecular modeling: TheE/DRY motif

A detailed analysis of the pharmacological properties of a b2-AR CAM wasinstrumental to propose the “allosteric ternary complex model” (Samamaet al., 1993). This extended version of the ternary complex model explicitlyrecognized the allosteric transition between at least two interconvertibleallosteric “states,” R (inactive or ground state) and R* (active). In theabsence of the agonist, R predominates, whereas agonists trigger the equi-librium toward R* thus favoring its stabilization. This analysis led to thesuggestion that constitutively activating mutations mimic, at least to someextent, the conformational change triggered by agonist binding to a wild-type GPCR.

It therefore made the hypothesis that, in the absence of agonist, struc-tural “constraints” keep the wild-type receptor in its ground or inactivestate (R), whereas agonist binding or activating mutations release suchconstraints triggering the conversion of the receptor into its active state(R*), which couples to G proteins. Once this hypothesis was made, wecombined 3-D model building of the a1b-AR structure with moleculardynamics (MD) simulations of the receptor to compare the structural/dynamic features of the wild-type structure (ground or inactive state) withthat of CAMs carrying mutations of A293 (Scheer et al., 1996).

The first a1b-AR model was built using an iterative ab initio procedureconsisting of upgrading and complicating the model to incorporate theincreasing number of experimental data on rhodopsin and the homologousGPCRs, as previously described (Fanelli et al., 1998). The ab initiomodel ofthe a1b-AR was extremely useful for developing a computational approachwhich allowed to define the structural/dynamic features differentiating theinactive from the active receptor states. These studies highlighted that thecationic residue R143(3.50) of the highly conserved E/DRY motif atthe cytosolic end of helix 3 (Fig. 7.1) undergoes the most conspicuouschanges. Whereas in the inactive state R143(3.50) is involved in persistenthydrogen bonding interactions with residues forming a highly conserved“polar pocket” (Scheer et al., 1996), the active states are characterized by aprogressive shift of R143(3.50) out of this pocket. The model predicted thatprotonation of the aspartate (D142(3.49)) of the E/DRYmotif would inducea conformational change of the receptor leading to the shift of R143(3.50)

out of the “polar pocket,” a hall mark of the receptor active states. Site-directed mutagenesis of D142(3.49) confirmed this prediction since


mutations of the aspartate generated a family of CAMs (Fig. 7.2; Scheeret al., 1996, 1997).

The interpretative and predictive abilities of the computational approachimproved as soon as the first crystal structure of rhodopsin became available,as extensively described elsewhere (Fanelli and De Benedetti, 2005).Despite the structural differences between the ab initio and comparativemodels, they both suggested that in the a1b-AR the highly conservedE/DRY motif is an important switch of receptor activation (Scheer et al.,2000). Increased constitutive activity was also found after mutating theacidic residue of the E/DRY motif in other receptors including rhodopsin(Cohen et al., 1993) and the b2-AR (Rasmussen et al., 1999). Altogether,these findings strongly suggest that mutations of the highly conservedE/DRY sequence might represent a general strategy to increase constitutiveactivity of GPCRs belonging to the rhodopsin-like class of receptors.

2.2. Microdomains involved in receptor activation

In the homology model of the a1b-AR (Greasley et al., 2002), the arginineof the E/DRY motif displays a double salt bridge with both the adjacentD142(3.49) and a glutamate (E289(6.30)) on the cytosolic end of helix 6(Fig. 7.1). This interaction pattern of R143(3.50), inherited from the rho-dopsin structure, constitutes an important feature of the ground or inactivestate of the receptor (Hofmann et al., 2009; Palczewski et al., 2000). Similarto the effect induced by mutating the aspartate of the E/DRY motif,mutations of E289(6.30) on helix 6 markedly increased the constitutiveactivity of the a1b-AR (Fig. 7.2; Greasley et al., 2002). Also mutations ofF6.44, belonging to an aromatic cluster on helix 6 modulating the helix3/helix 6 packing, increased the constitutive activity of the a1b-AR.

These findings provided a strong basis in favor of a theoretical model ofreceptor activation in which the E/DRY microdomain is part of a networklinking helices 3 and 6—the so-called helix 3/helix 6 “ionic lock”(Hofmann et al., 2009). Whereas this “ionic lock” is a fundamental struc-tural constraint keeping the receptor inactive, the release or weakening ofthe interactions involving R143(3.50) results in receptor activation. It is nowwell established that this putative model of receptor activation is shared byother GPCRs belonging to the rhodopsin-like class, as recently reviewed(Gether, 2000).

Recent results on rhodopsin structures indicate that the NPXXY(X)5,6Fon helices 7 and 8 (Fig. 7.1) is another microdomain which, in concert withthe E/DRY motif, controls the structural changes underlying photorecep-tor activation (Hofmann et al., 2009). In particular, the NPXXY(X)5,6Fmotif provides two constraints: one, involving N(7.49), is represented by theH-bonding network linking helices 1, 2, and 7, and the other, involvingY(7.53) and F(7.60), establishes a link between helices 7 and 8.


These interactions, which might be conserved in different GPCRs, couldbe interesting targets of site-directed mutagenesis studies to generate CAMs.

2.3. Constitutively activating mutations of the a1a- anda1b-AR subtypes

Combining molecular modeling and site-directed mutagenesis, our grouphas found a number of activating mutations in the human a1a and in thehamster a1b-AR subtypes. CAMs of the a1dAR subtype have not beenreported so far. In our studies, constitutive activity was mainly assessedmeasuring IP accumulation in whole cells, as described in Section 3.

Most of the activating mutations are predicted to perturb the helix3/helix 6 packing of the receptor (Figs. 7.1 and 7.2). These include thefollowing findings:

(i) All possible amino acid substitutions of A293(6.34) in the cytosolicextension of helix 6 in the a1b-AR induced variable levels of constitu-tive activity (Kjelsberg et al., 1992); the homologous mutation ofA271(6.34) in the a1a-AR into glutamate or lysine also increases theconstitutive activity of the receptor (Rossier et al., 1999).

(ii) All possible natural amino acid substitutions of D142(3.49) of theE/DRY motif in the a1b-AR resulted in variable levels of agonist-independent activity (Scheer et al., 1997); CAMs displayed increasedaffinity for the full agonist epinephrine which was, at least to someextent, correlated with their degree of constitutive activity; mutatingthe homologous D123(3.49) into isoleucine in the a1a-AR activated thereceptor (Rossier et al., 1999).

(iii) In the a1b-AR mutations of E289(6.30), at the cytosolic end of helix,into A, D, F, K, and R resulted in a marked increase in the constitutiveactivity of the receptor as well as in its affinity for epinephrine(Greasley et al., 2002); these mutations are predicted to release the“ionic lock” linking helices 3 and 6.

(iv) Replacement of the highly conserved F303(6.44) in helix 6 of the a1b-AR with leucine also resulted in increased constitutive activity(Greasley et al., 2002).

Interesting findings were obtained by mutating the arginine of theE/DRY sequence of the a1b-AR into K, H, D, E, A, I, and N (Scheeret al., 2000). The charge-conserving mutation of R143(3.50) into lysine andhistidine conferred, respectively, high and low degree of constitutive activ-ity to the receptor. In contrast, all the other replacements of R143(3.50) werenot constitutively active and were dramatically impaired in their ability tomediate agonist-induced IP response.

Few other activating mutations were found by other groups in theextracellular half of the seven-helix bundle: mutations of C128(3.35) on


helix 3 (Porter and Perez, 1999) and of A204(5.39) on helix 5 of the a1b-AR(Hwa et al., 1996), mutations of M292 in the rat a1a-AR (Hwa et al., 1996).

3. Measuring Constitutive Activity of

Receptor-Mediated Gq Activation

3.1. Constitutive activity of a1-AR CAMs

In our studies, constitutive activity of the a1-AR CAMs was mainlyassessed measuring agonist-independent accumulation of IPs in wholecells (COS-7 or HEK293) expressing the recombinant receptors. Experi-ments measuring constitutive activity in whole cells have the main advan-tage of amplifying the receptor output signal in experiments measuringthe accumulation of second messengers under conditions in which theirdegradation is inhibited. However, their results should be interpreted withcaution for several reasons including the presence of endogenous ligandsactivating the receptor or receptor regulatory events (e.g., desensitizationor upregulation).

A crucial problem in experiments measuring constitutive activity inwhole cells concerns the relationship between the receptor activity andthe expression levels of different receptors. As predicted by the “allostericternary complex model” (Samama et al., 1993), the constitutive activitymeasured in cells depends on the amount of expressed receptor in its activeform. Therefore, to compare the constitutive activity of wild-type andmutated receptors, these should be expressed at the same level, which isoften difficult to achieve in transfected cells. Two main approaches havebeen used to overcome this problem.

In most studies on CAMs of the a1bAR, different amounts of receptorDNA have been transfected to obtain similar expression levels (Scheeret al., 1996), as shown in Fig. 7.3. In these studies, the constitutive activitycould be directly compared among different receptors in the same experi-ment. In other studies, the constitutive activity of different receptors wasnormalized to their receptor number after assessing the existence of alinear relationship between constitutive activity and receptor number foreach receptor (Rossier et al., 1999). Both methods rely on the carefulmeasurement of receptor expression. In fact, if the expression level of areceptor is underestimated, its constitutive activity might be overesti-mated. This could happen for receptor mutants expressed at levels toolow to obtain reliable binding data or displaying changes in the affinity forthe ligand. In the vast majority of studies on CAMs, receptor expressionhas been measured using ligand binding. It might be useful in some casesto monitor the expression of the protein by Western blotting to confirmthe ligand-binding data.

10

8

6

4

2

0

0 0.80.4 1.2 1.6

Bas

al I

P (

fold

abo

ve c

ontr

ol)

Receptor density (pmol/mg protein)

D142A

WT

Figure 7.3 Relationship between constitutive activity and receptor expression. Celltransfection and total inositol phosphate (IP) determination was as described in Fig. 7.2.Membrane preparations derived from cells expressing the different receptors andligand-binding experiments using [125I]HEAT were performed as described elsewhere(Cotecchia et al., 1992). COS-7 cells were transfected with increasing amounts of DNAwhich ranged from 0.2 to 2.0 mg/million of cells for the wild type and from 1.0 to6.0 m/million of cells for D142A, each performed in triplicate. Basal IPs are expressed aspercentage above control which indicates cells not expressing the receptors. The resultsare from several independent experiments.


To overcome these problems, it would be ideal to measure constitutiveactivity in membrane assays or directly monitoring receptor-mediated Gprotein activation.

For GPCRs coupled to Gi, receptor-mediated binding of [35S]GTPgS toGai in membrane preparations has been successfully used to measure consti-tutive activity. However, for GPCRs linked to the Gs or Gq/11 signalingpathway receptor-mediated binding of [35S]GTPgS gives smaller signalsprobably because of the lower abundance of the G proteins in the cells.

To detect constitutive activity of the a1b-AR and its CAMs measuringguanine nucleotide exchange, the receptors have been fused to Ga11subunit and [35S]GTPgS binding was measured in membranes from cellsin which these constructs were overexpressed (Carrillo et al., 2002). Toincrease the signal, the radiolabeled Ga11 was immunoprecipitated withantibodies specific for its C-tail. The results from this study showed thatagonist-independent [35S]GTPgS binding was significantly greater at theCAMs compared to the wild-type a1b-AR. Another approach could be tocoexpress receptors with Gaq or 11 and immunoprecipitate the [35S]GTPgS bound. These approaches are certainly useful to demonstrate con-stitutive activity in a cell-free system, but they are more time consuming andless sensitive compared to assays in intact cells.


CAMs of the AR display also a number of interesting features that couldbe used to define the profile of constitutively active receptors: increasedbinding affinity for agonists (Samama et al., 1993; Scheer et al., 1997),greater efficacy of partial agonists (Samama et al., 1993), increased basalphosphorylation (Pei et al., 1994; Cotecchia & Mhaouty-Kodja, 1999),enhanced endocytosis (Mhaouty-Kodja et al., 1999). However, these prop-erties differ among receptor mutants depending also on the localization ofthe mutations and cannot be generalized to all CAMs.

3.2. Constitutive activity of wild-type a1-ARs

The “allosteric ternary complex model” predicts that the constitutive activ-ity measured in cells depends on the amount of expressed receptor in itsactive stateR* (Samama et al., 1992). This prediction is clearly supported byfindings demonstrating that increasing density of GPCRs results in theprogressive elevation of basal (agonist-independent) receptor-mediatedproduction of second messengers. This implies also that native GPCRsmight display spontaneous activity in physiological systems which hasbeen demonstrated only for few receptors. There are documented differ-ences in the extent of constitutive activity among even highly relatedGPCRs and this might depend on differences in their intrinsic activationproperties, on the cellular environment in which signaling is measured aswell as on experimental factors.

For the a1-AR subtypes, we reported constitutive activity of thewild-type a1a- and a1b-AR when the receptors were overexpressed inCOS-7 cells (Rossier et al., 1999). Constitutive or basal activity of thewild-type receptor was assessed comparing the IP accumulation intransfected cells with that of mock transfected cells. The spontaneousactivity of the a1b was greater than that of the a1a-AR expressed atsimilar levels (2–3 pmol/mg of protein) (Fig. 7.4). Constitutive activityof the a1a- or a1b-AR in physiological systems has not been investigated,whereas studies on the a1d-AR are summarized in another chapter of thisvolume. This does not exclude that native a1-AR subtypes might havesome constitutive activity which could fulfill specific roles in signaling. Suchissues should be further explored and the elucidation of their physiologicalimplications might represent an important area of investigation.

4. Inverse Agonism at the a1-ARs

Negative efficacy, that is, the capacity of an antagonist binding to itsreceptor to repress its spontaneous activity, has been a concept implicit inreceptor theory from the start (Costa and Cotecchia, 2005). However, it

aa

1000

2500

2000

1500

1000

500

2000

3000

4000

5000

0 0Con a1b-ARa1a-ARCon

Bas

al [

3 H]IP (

cpm

/dish)

Bas

al [

3 H]IP (

cpm

/dish)

Figure 7.4 Constitutive activity of the wild-type a1a- and a1b-AR. Total inositolphosphate (IP) accumulation was measured, as described in Fig. 7.2, in control COS-7 cells (Con) not expressing the receptors or in cells expressing the wild-type a1a- ora1b-AR. Basal IPs were measured after 100 min incubation in the absence of ligands.Receptor expression ranged 2–3 pmol/mg of proteins. Statistical significance wasanalyzed by unpaired Student’s t test a, P < 0.05 Bas of cells expressing the receptorversus control cells. The figure is from Rossier et al. (1999).


remained an undeveloped idea for many years largely because of thedifficulty to find adequate experimental systems to test it. The availabilityof CAMs and of cell systems overexpressing wild-type GPCRs represents auseful tool to identify drugs with negative efficacy which is commonlymeasured as their ability of inhibiting the agonist-independent receptoreffect. Ligands displaying negative efficacy have been indicated with theterm of inverse agonists or negative antagonists without any specific refer-ence to the mechanistic basis of their effect which might differ amongligands.

To identify inverse agonists at the a1-AR subtypes, we tested 24 alpha-antagonists differing in their chemical structures for their capacity todecrease the basal activity of CAMs of the a1a- and a1b-AR subtypes(Fig. 7.5; Rossier et al., 1999). The receptors were expressed in COS-7cells and constitutive activity was assessed measuring agonist-independentaccumulation of IPs. Drugs, previously characterized for their bindingaffinity, were tested at saturating concentrations.

As shown in Fig. 7.5, the vast majority of alpha-antagonists displayedinverse agonism. However, the various alpha-antagonists differed in theirnegative efficacy (varying from about 20% to 90% of inhibition of basalreceptor response) and some of these differences depended on thea1-AR subtype. In fact, a large number of structurally different alpha-antagonists including all the tested quinazolines were inverse agonists at

100

100

50

50

0

0

A271E

A293E

5-M

ethy

lura

pidi

lB

MY

737

8R

EC

15/

3039

RE

C 1

5/27

39R

EC

15/

2869

RE

C 1

5/30

11W

AY

100

635

SNA

P 5

089

S(+

)-ni

guld

ipin

e

Phe

ntol

amin

e

WB

410

1B

E 2

254

Tam

sulo

sin

RS-

1705

3

Pra

zosin

Ter

azos

in(+

)-cy

claz

osin

(–)-

cycl

azos

inR

EC

15/

2615

Alfuz

osin

Indo

ram

inC

oryn

anth

ine

Spip

eron

eA

H11

110A

5-M

ethy

lura

pidi

lB

MY

737

8R

EC

15/

3039

RE

C 1

5/27

39R

EC

15/

2869

RE

C 1

5/30

11W

AY

100

635

S(+

)-ni

guld

ipin

eSN

AP 5

089

Phe

ntol

amin

e

WB

410

1B

E 2

254

Tam

sulo

sin

RS-

1705

3

Pra

zosin

Ter

azos

in(+

)-cy

claz

osin

(–)-

cycl

azos

inR

EC

15/

2615

Alfuz

osin

Indo

ram

inC

oryn

anth

ine

Spip

eron

eA

H11

110A

[3H

]IP (

% o

f ba

sal)

[3H

]IP (

% o

f ba

sal)

Figure 7.5 Inverse agonists at constitutively active a1-AR mutants. Total inositolphosphate (IP) accumulation was measured, as described in Fig. 7.2, in COS-7 cellsexpressing the a1a-AR CAM, A271E, or a1b-AR CAM, A293E, in the absence (basal)or presence of different ligands at a concentration of 10�5

M (10�4M for REC

15/3039) for 45 min. The ligands are grouped according to their structural similarities(from left to right the groups include N-arylpiperazines; 1,4-dihydropyridines; imida-zolines; benzodioxanes and phenylalkylamines; quinazolines; various structures). Theresults are expressed as % of basal which indicates the basal levels of IP measured inuntreated cells. Results are the mean � S.E. of three to six independent experiments.The figure is from Rossier et al. (1999).


both the a1a- and a1b-AR subtypes. In contrast, several N-arylpiperazinesdisplayed different properties at the two a1-AR subtypes being inverseagonists with profound negative efficacy at the a1b-AR, but not at thea1a. To assess whether the effect of the alpha-blockers on CAMs reflectedtheir activity of wild-type receptors, few ligands were tested also for their


capacity to inhibit the basal activity of the wild-type a1a- and a1b-AR.These findings indicated that the behavior of the drugs was similar at CAMor wild-type receptor suggesting that the use of CAMs is a good tool toscreen for inverse agonists.

An important problem in experiments measuring inverse agonism inwhole cells is to confirm that the effect of the ligand is mediated by thereceptor and not by other unknown mechanisms on signaling. For thispurpose, we further investigated the effect on the a1-AR CAMs of twoligands, prazosin and 5-methylurapidil, the first being almost a full inverseagonist and the second having only modest negative efficacy. The inhibitionof basal IP accumulation by prazosin was competitively inhibited by increas-ing concentrations of 5-methylurapidil thus confirming that the inhibitoryeffect of the inverse agonist prazosin was mediated by the receptor (Rossieret al., 1999).

Studies on the structure–activity relationship of alpha-blockers differingin their negative efficacy in combination with molecular modeling of thereceptors might help delineating receptor domains crucially involved in theinhibition of receptor isomerization and activation.

The therapeutic benefit of inverse agonists in diseases related to sponta-neous activating mutations of GPCRs is quite obvious. However, a ques-tion which remains to be answered is whether therapeutic differences andbenefits exist in the clinical use of drugs having negative efficacy versusthose that behave as neutral blockers at native receptors. With respect to thisquestion, it is important to highlight that drugs with different degrees ofnegative efficacy might differ in their ability to induce upregulation ofGPCRs upon chronic treatment (Lee et al., 1997). Answering tomany open questions about the therapeutic implications of inverse agonismwill remain an interesting and important area of investigation in thenext years.

5. Conclusions

The studies on CAMs of the a1AR subtypes and of other GPCRs hadan important impact on our understanding of the molecular mechanismsunderlying GPCR activation and inverse agonism. Structural information athigh resolution on other GPCRs than rhodopsin will be necessary toimprove our understanding of GPCR activation and drug action at amolecular level. In addition, it will be important to understand the implica-tions of drugs with negative efficacy in vivo, evaluate their benefits andimprove future therapeutic strategies.


ACKNOWLEDGMENTS

The work in S. Cotecchia’s laboratory was supported by the Fonds National Suisse de laRecherche Scientifique (grant no. 31000A0-10073).

REFERENCES

Ballesteros, J. A., and Weinstein, H. (1995). Integrated methods for the construction ofthree dimensional models and computational probing of structure-function relations inG-protein coupled receptors. Methods Neurosci. 125, 366–428.

Carrillo, J. J., Stevens, P. A., and Milligan, G. (2002). Measurement of agonist-dependent andindependent signal initiation of alpha(1b)-adrenoceptor mutants detected by direct analysis ofguanine nucleotide exchange on the G protien-galpha(11). J. Pharmacol. Exp. Ther. 302,1080–1088.

Cavalli, A., Fanelli, F., Taddei, C., De Benedetti, P. G., and Cotecchia, S. (1996). Aminoacids of the alpha1B-adrenergic receptor involved in agonist binding: Differences indocking catecholamines to receptor subtypes. FEBS Lett. 399, 9–13.

Cohen, G. B., Yang, T., Robinson, P. R., and Oprian, D. D. (1993). Constitutive activationof opsin: Influence of charge at position 134 and size at position 296. Biochemistry 32,6111–6115.

Costa, T., and Cotecchia, S. (2005). Historical review: Negative efficacy and the constitutiveactivity of G-protein coupled receptors. Trends Pharmacol. Sci. 26, 618–624.

Cotecchia, S., and Mhaouty-Kodja, S. (1999). Regulatory mechanisms of alpha1b-adrenergicreceptor function. Biochem. Soc. Trans. 27, 154–157.

Cotecchia, S., Exum, S., Caron, M. G., and Lefkowitz, R. J. (1990). Regions of alpha1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysisand enhanced sensitivity of biological function. Proc. Natl. Acad. Sci. USA 87,2896–2900.

Cotecchia, S., Ostrowski, J., Kjelsberg, M. A., Caron, M. G., and Lefkowitz, R. J. (1992).Discrete amino acid sequences of the a1-adrenergic receptor determine the selectivity ofcoupling to phosphatidylinositol hydrolysis. J. Biol. Chem. 267, 1633–1639.

Diviani, D., Lattion, A. L., Larbi, N., Kunapuli, P., Pronin, A., Benovic, J. L., andCotecchia, S. (1996). Effect of different G protein-coupled receptor kinase- and proteinkinase C-mediated desensitization of the alpha1B-adrenergic receptor. J. Biol. Chem.271, 5049–5058.

Diviani, D., Lattion, A. L., and Cotecchia, S. (1997). Characterization of the phosphoryla-tion sites involved in G protein-coupled receptor kinase- and protein kinase C-mediateddesensitization of the alpha1B-adrenergic receptor. J. Biol. Chem. 272, 28712–28719.

Fanelli, F., and De Benedetti, P. G. (2005). Computational modeling approaches tostructure–function analysis of G protein-coupled receptors. Chem. Rev. 105, 3297–3351.

Fanelli, F., Menziani, C., Scheer, A., Cotecchia, S., and De Benedetti, P. G. (1998). Abinitio modeling and molecular dynamics simulation of the alpha1b-adrenergic receptoractivation. Methods 14, 302–317.

Fanelli, F., Menziani, C., Scheer, A., Cotecchia, S., and De Benedetti, P. G. (1999).Theoretical study on the electrostatically driven step of receptor–G protein recognition.Proteins Struct. Funct. Genet. 37, 145–156.

Gether, U. (2000). Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev. 21, 90–113.

Greasley, P. J., Fanelli, F., Scheer, A., Abuin, L., Nenniger-Tosato, M., De Benedetti, P. G.,and Cotecchia, S. (2001). Mutational and computational analysis of the alpha1b-


adrenergic receptor: Involvement of basic and hydrophobic residues in receptor activa-tion and G protein coupling. J. Biol. Chem. 276, 46485–46494.

Greasley, P., Fanelli, F., Rossier, O., Abuin, L., and Cotecchia, S. (2002). Mutagenesis andmodelling of the alpha1b-adrenergic receptor highlights the role of the helix 3/helix 6interface in receptor activation. Mol. Pharmacol. 61, 1025–1032.

Hofmann, K. P., Scheerer, P., Hildebrand, P. W., Choe, H.-W., Park, J. H., Heck, M., andErnst, O. P. (2009). A G protein-coupled receptor at work: The rhodopsin model. TrendsBiochem. Sci. 34, 540–552.

Hwa, J., and Perez, D. M. (1996). Identification of critical determinants of alpha1-adrenergicreceptor subtype selective agonist binding. J. Biol. Chem. 271, 6322–6327.

Hwa, J., Graham, R. M., and Perez, D. M. (1996). Chimeras of alpha1-adrenergic receptorsubtypes identify critical residues that modulate active state isomerization. J. Biol. Chem.271, 7956–7964.

Kjelsberg, M. A., Cotecchia, S., Ostrowski, J., Caron, M. G., and Lefkowitz, R. J. (1992).Constitutive activation of the alpha1B-adrenergic receptor by all amino acid substitutionat a single site. J. Biol. Chem. 267, 1430–1433.

Lattion, A. L., Diviani, D., and Cotecchia, S. (1994). Truncation of the receptor impairsagonist-induced phosphorylation and desensitization of the alpha1B-adrenergic receptor.J. Biol. Chem. 269, 22887–22893.

Lee, T. W., Cotecchia, S., and Milligan, G. (1997). Up-regulation of the levels of expressionand function of a constitutively active mutant of the hamster alpha1B-adrenoceptor byligands that act as inverse agonists. Biochem. J. 325, 733–739.

Mhaouty-Kodja, S., Barak, L. S., Scheer, A., Abuin, L., Diviani, D., Caron, M. G., andCotecchia, S. (1999). Constitutively active alpha1b-adrenergic receptor mutants displaydifferent phosphorylation and internalization properties. Mol. Phamacol. 55, 339–347.

Palczewski, C., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., LeTrong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M.(2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289,739–745.

Pei, G., Samama, P., Lohse, M., Wang, M., Codina, J., and Lefkowitz, R. J. (1994).A constitutively active mutant b2-adrenergic receptor is constitutively desensitized andphosphorylated. Proc. Natl. Acad. Sci. USA 91, 2699–2702.

Porter, J. E., and Perez, D. M. (1999). Characteristics for a salt-bridge switch mutation of thealpha(1b) adrenergic receptor. Altered pharmacology and rescue of constitutive activity.J. Biol. Chem. 274, 34535–34538.

Rasmussen, S. G. F., Jensen, A. D., Liapakis, G., Ghanouni, P., Javitch, J. A., and Gether, U.(1999). Mutation of highly conserved aspartic acid in the beta2 adrenergic receptor:Constitutive activation, structural instability and conformational rearrangement of trans-membrane segment 6. Mol. Pharmacol. 56, 175–184.

Ren, Q., Kurose, H., Lefkowitz, R. J., and Cotecchia, S. (1993). Constitutively activemutants of the a2A-adrenergic receptor. J. Biol. Chem. 268, 16483–16487.

Rossier, O., Abuin, L., Fanelli, F., Leonardi, A., and Cotecchia, S. (1999). Inverse agonismand neutral antagonism at alpha(1a)- and alpha(1b)-adrenergic receptor subtypes. Mol.Pharmacol. 56, 858–866.

Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993). A mutation-inducedactivated state of the beta2-adrenergic receptor: Extending the ternary complex model.J. Biol. Chem. 268, 4625–4636.

Scheer, A., Fanelli, F., Costa, T. De., Benedetti, P. G., and Cotecchia, S. (1996). Constitu-tively active mutants of the alpha1B-adrenergic receptor: Role of highly conserved polaramino acids in receptor activation. EMBO J. 15, 3566–3578.


Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G., and Cotecchia, S. (1997). Theactivation process of the alpha1B-adrenergic receptor: Potential role of protonation andhydrophobicity of a highly conserved aspartate. Proc. Natl. Acad. Sci. USA 94, 808–813.

Scheer, A., Costa, T., Fanelli, F., De Benedetti, P. G., Mhaouty-Kodja, S., Abuin, L.,Nenniger-Tosato, M., and Cotecchia, S. (2000). Mutational analysis of the highlyconserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergicreceptor: Effects on receptor isomerization and activation. Mol. Pharmacol. 57, 219–231.

Schwinn, D. A., Johnston, G. I., Page, S. O., Mosley, M. J., Wilson, K. H., Worman, N. P.,Campbell, S., Fidock, M. D., Furness, L. M., Parry-Smith, D. J., Beate, P., andBailey, D. S. (1995). Cloning and pharmacological characterization of human alpha-1adrenergic receptors: Sequence corrections and direct comparison with other specieshomologues. J. Pharmacol. Exp. Ther. 272, 134–142.

Vazquez-Prado, J., Medina, L. C., Romero-Avila, M. T., Gonzalez-Espinosa, C., andGarcia-Sainz, J. A. (2000). Norepinephrine- and phorbol ester-induced phosphorylationof alpha(1a)-adrenergic receptors. Functional aspects. J. Biol. Chem. 275, 6553–6559.

[methods in enzymology] constitutive activity in receptors and other proteins, part b volume 485 ||...

Documents