Báo cáo khoa học: Allosteric functioning of dimeric class C G-protein-coupled receptors doc

Activation mechanism of class C GPCRs involves allosteric interaction between the VFTs As described above, the mGlu1 VFT can reach a closed state stabilized by agonists, and form dimers

Allosteric functioning of dimeric class C G-protein-coupled receptors

J-P Pin1–5, J Kniazeff1–5, J Liu1–5, V Binet1–5, C Goudet1–5, P Rondard1–5and L Pre´zeau1–5

1 Institut de Ge´nomique Fonctionnelle, Montpellier, France

2 CNRS, UMR5203, Montpellier, France

3 INSERM, Montpellier, France

4 Universite´ Montpellier-I, France

5 Universite´ Montpellier-II, France

Most membrane receptors, including ligand-gated

channels, tyrosine kinase receptors, cytokine receptors

and guanylate cyclase receptors form oligomers This

was rapidly recognized as being crucial for the

func-tioning of these receptors In the case of ligand-gated

channel receptors, association of 4–5 subunits is

required to form an ion channel In the case of

recep-tors that have a single transmembrane domain, it was

difficult to imagine how the signal could be transduced

from the extracellular to the intracellular side of the

membrane without subunit association In that case, it

was rapidly proposed that ligand binding in the

extra-cellular domain induces receptor dimerization, allowing

the associated intracellular enzymatic domains to

inter-act and become inter-activated More recent data from the

determination of the three-dimensional structure of the

extracellular domains of such receptors with and with-out agonists, revealed that they can even be consti-tutive dimers, agonists stabilizing a specific active conformation of the dimer [1,2]

In contrast, all G-protein-coupled receptors (GPCRs) have a large membrane core domain com-posed of seven transmembrane-spanning helices, which

is responsible, in most cases, for both ligand recogni-tion and activarecogni-tion of the intracellular effector, i.e the heterotrimeric G-protein This, plus other biophysical data, lead to the conclusion that GPCRs work as monomers that can oscillate between various confor-mations, the active conformations being stabilized by agonists, whereas the fully inactive conformations are stabilized by inverse agonists However, it was difficult

to explain some cooperativity phenomena observed in

Keywords

activation mechanism; allosteric modulators;

dimerization; GPCR

Correspondence

J-P Pin, Institut de Ge´nomique

Fonctionnelle, 141 rue de la Cardonille,

F-34094 Montpellier cedex 5, France

Fax: +33 467 54 2432

Tel: +33 467 14 2988

E-mail: jppin@ccipe.cnrs.fr

(Received 16 February 2005, accepted

6 April 2005)

doi:10.1111/j.1742-4658.2005.04728.x

Whereas most membrane receptors are oligomeric entities, G-protein-coupled receptors have long been thought to function as monomers Within the last 15 years, accumulating data have indicated that G-protein-coupled receptors can form dimers or even higher ordered oligomers, but the gen-eral functional significance of this phenomena is not yet clear Among the large G-protein-coupled receptor family, class C receptors represent a well-recognized example of constitutive dimers, both subunits being linked, in most cases, by a disulfide bridge In this review article, we show that class C G-protein-coupled receptors are multidomain proteins and highlight the importance of their dimerization for activation We illustrate several consequences of this in terms of specific functional properties and drug development

Abbreviations

Acc, active-closed-closed conformation; Aco, active-closed-open conformation; CaS, receptor, calcium-sensing receptor; CRD, cystein-rich domain; ER, endoplasmic reticulum; HD, heptahelical domain; mGlu, receptor, metabotropic glutamate receptor; Roo, resting-open-open conformation; T1R: taste receptor type 1; VFT, Venus flytrap domain.

ligand binding This led to the demonstration that

most GPCRs can oligomerize as shown by both

bio-chemical and energy transfer technologies [3] In recent

years, several publications have indicated that this

phe-nomenon is involved in trafficking of the receptor to

and from the plasma membrane, and in specific

cross-talk between receptor subtypes [4] However, the

pre-cise role and importance of GPCR oligomerization in

the activation process remains unknown

Five main classes of GPCRs can be defined in

mam-mals based on sequence similarity [5–7] Whereas the

large number of rhodopsin-like receptors form class A,

secretin-like and metabotropic glutamate (mGlu)-like

receptors are members of classes B and C, respectively

Frizzled receptors and a subgroup of pheromone

receptors form two additional classes Class C GPCRs

have been shown to be constitutive dimers and therefore

represent a good model for studying the functional

rele-vance of GPCR dimerization These receptors include

those for the main neurotransmitters, glutamate and

GABA, as well as a receptor activated by extracellular

Ca2+, some pheromone receptors and receptors for the

sweet and umami taste compounds [8] In this review

article, we summarize our knowledge on the functioning

of class C GPCRs and illustrate how allosteric

inter-actions between the subunits play a fundamental role

in their activation Of interest, we see that this

com-plex functioning of class C receptors offers a number

of possibilities to regulate their activity with synthetic

ligands acting at sites different from the natural

ligand-binding site, the so-called allosteric modulators

The multiple domains of class C GPCRs

In contrast to most class A rhodopsin-like GPCRs,

class C receptors are composed of three main

struc-tural domains, not including the C-terminal tail which

can be very long (up to 376 residues for mGlu5b) and

where a multitude of intracellular scaffolding and

sig-nalling molecules bind These domains are the Venus

flytrap domain (VFT), which contains the

agonist-binding site, the cysteine-rich domain (CRD) and the

heptahelical domain (HD) involved in G-protein

acti-vation (Fig 1)

The VFT module is a bilobate domain that shares

structural similarity with bacterial periplasmic amino

acid-binding proteins The structure of the mGlu1

VFT has been solved by X-ray crystallography in the

absence and presence of either agonist or antagonist

[9,10] These studies revealed that both types of ligand

bind in the cleft that separates both lobes As already

shown for bacterial proteins, these studies also

revealed that the VFT of class C GPCRs can adopt

either an open or a closed conformation (Fig 1) Inter-estingly, both conformations have been seen in the absence of ligand, as well as in the presence of agon-ists In contrast, only the open conformation was observed with bound antagonist It was therefore pro-posed that the VFT can naturally oscillate between these two states, the closed state being stabilized by agonists, whereas antagonists prevent the closure Further studies performed on full-length receptors confirmed this functioning of the VFT For example, by removing steric or ionic hindrance that prevents mGlu8 VFT closing upon antagonist binding, two antagonists were converted into full agonists [11] Moreover, the introduction of two cysteine residues that are expected, based on modelling studies, to cross-link both lobes of the GABAB1 receptor and lock it in a closed state, generates a fully constitutively active receptor [12] The CRD links the VFT to the HD in most class C GPCRs The structure of this CRD is not known although a three-dimensional model has been proposed recently [13] (Fig 1) Although the CRD is absent in the GABAB receptor subunits, it appears necessary for the activation of either mGlu or calcium-sensing (CaS) receptors [14], but its specific mode of action is not yet known

Like any other GPCRs, class C receptors possess a

HD that shares very low sequence similarity with rho-dopsin-like receptors (Fig 1) Indeed, few residues are conserved in these two groups of receptors and model-ling studies suggest that both types of HD share a similar structure [8] As in class A receptors, the intra-cellular loops of class C GPCRs as well as the C-terminal tail are involved in G-protein coupling For various class C GPCRs, including the mGlu5, GABAB2and CaS receptors, the HD can fold correctly and be trafficked to the cell surface when expressed alone after deletion of both the large extracellular domain and the long C-terminal tail [15–17] More-over, these isolated HDs retain their ability to activate G-proteins as illustrated by their constitutive activity,

an activity that can either be inhibited by inverse agon-ists known to bind in the HD, or further stimulated by other molecules known as positive allosteric modula-tors Accordingly, the HD of class C GPCRs appears

to behave like rhodopsin, oscillating between various states each being possibly stabilized by specific com-pounds (Fig 1)

In summary, class C GPCRs are multimodule pro-teins and both major modules (the agonist-binding VFT and the G-protein-activating HD) retain their specific functional properties when isolated As expec-ted for allosteric proteins, these modules can oscillate between various states, each being stabilized by specific

molecules However, how can the ligand-binding

domain control the activity of the HD? In other

words, how is the signal transduced from one domain

to the other?

Class C GPCRs are constitutive dimers

An important piece of information to understand the

activation process of class C GPCRs came with the

discovery that these receptors are constitutive dimers

The first observation came from the mGlu5 receptor,

which was shown in western blot and

immunopreci-pitation experiments to be a homodimer in both

transfected cells and native tissue [18] Only upon

di-thiothreitol treatment was the monomeric form

detec-ted Soon after, the CaS receptor was also shown to

form dimers stabilized by a disulfide bridge via Cys129

located in the VFT [19], and this was confirmed in

both mGlu1 and mGlu5 receptors [20] Because this residue is conserved in all mGlu receptors, as well as

in the taste and pheromone receptors, these are also expected to be disulfide-linked dimers Mutation of this Cys residue does not prevent dimer formation [21] Indeed, the VFT, even when produced as a soluble protein, forms stable dimers via a hydrophobic surface area located on one side of lobe-I, as clearly revealed

in the crystal structure of the dimers of mGlu1 VFTs [9,10] (Fig 2A) Mutation of the Cys residue involved

in the covalent linkage of the subunit also does not affect functioning of the receptor [22] Although the role of this disulfide bridge remains elusive, it certainly prevents any possible dissociation of the subunits under normal conditions, making these receptors con-stitutive dimers

To date, no heterodimeric mGlu receptors have been described Only mGlu1–CaS heterodimers have been

VFT

CRD

HD

HD*

HD HDg

Fig 1 The main domains of class C GPCRs

and their various conformational states.

Class C GPCRs are composed of three main

structural domains, the Venus flytrap

domain (FVT) where agonists and

competit-ive antagonists bind, the cysteine-rich

domain (CRD) that interconnects the VFT to

the heptahelical domain (HD), and HD,

which if similar to rhodopsin-like GPCRs.

Each structural domain is shown in a ribbon

view Both the VFT and HD are coloured

according to the succession of secondary

structure elements from dark blue

(N-termi-nus) to red (C-termi(N-termi-nus) Both the open

unliganded and agonist-bound closed

confor-mation of the VFT are shown The three

expected conformational states for the HD

are indicated, as also proposed for the

rhodopsin-like GPCRs: HDg, ground totally

inactive state; HD, basal state; HD*, fully

active state The ribbon views were

gener-ated using the coordinates of the mGlu1

VFT (protein data bank Accession nos

1EWT:A and 1EWK:A, respectively), the

pro-posed model of the CRD, and the

coordi-nates of rhodopsin (protein data bank

Accession no 1F88).

observed [23], but more work is required to validate

their functional and physiological relevance However,

the related taste receptors need to heterodimerize to

form functional receptors The association of taste

receptor type 1 (T1R1) and taste receptor type 3

(T1R3) results in the formation of umami receptors

[24], whereas taste receptor type 2 (T1R2) and T1R3

constitute the sweet receptors [25] Although not

observed in heterologous expression systems, T1R3

may also be able to form a functional low-affinity

sweet receptor in the absence of T1R1 and T1R2 [26]

In contrast to the other class C GPCRs, the GABAB

receptor is not a disulfide-linked dimer However, this

receptor was the first GPCR identified as an obligatory

heterodimer composed of two distinct subunits,

GABAB1 and GABAB2 [27] During evolution, a sys-tem has been selected to ensure that only the func-tional heterodimer reaches the cell surface Indeed, the GABAB1 subunit contains an endoplasmic reticulum (ER) retention signal in its intracellular tail, preventing

it from reaching the surface alone [28] Only when associated with GABAB2 can this subunit reach the cell surface and be functional Although no covalent linkage between the subunits has been observed, these dimers are likely very stable due to a coiled coil inter-action at the level of their intracellular tail, as well as

by direct interaction of their VFTs and also likely their HDs [29]

These observations revealed that class C GPCRs are complex multidomain molecules and raised an

Lobe-I VFTs

Lobe-II

HDs

A

B

Fig 2 General structure of dimeric class C GPCRs (A) Ribbon view of the crystal struc-ture of the resting Roo (left, pdb Accession

no 1EWT) and fully active Acc (right, pdb Accession no 1ISR) state of the mGlu1 VFT dimer, and apposition of two rhodopsin structures The yellow subunit is in the front, whereas the blue subunit is in the back Note the difference in the relative ori-entation of the two VFTs probably leading to

a different mode of association of the two HDs within the dimer (B) Scheme illustra-ting that agonist binding in one VFT can activate the HD of the same subunit (cis-activation) and ⁄ or the HD of the other subunit (trans-activation) In the wild-type heterodimeric GABABreceptor only trans-activation occurs (agonist binding in the GABA B1 VFT leads to the activation of the GABAB2HD), but both cis- and trans-activa-tion occur in the homodimeric mGlu recep-tors.

ant issue: the interplay between the various states of

each domain in the dimer, and how this can be

con-trolled by agonists

Activation mechanism of class C

GPCRs involves allosteric interaction

between the VFTs

As described above, the mGlu1 VFT can reach a

closed state stabilized by agonists, and form dimers via

a hydrophobic area on one side of its lobe-I [9,21]

This contact between the VFTs is likely required for

receptor activation, because a point mutation in that

area results in a loss of function of the receptor, even

though agonist binding can still be measured [30]

Comparison of the crystal structure of the VFT dimer

in the absence or presence of glutamate also revealed a

major change in the relative orientation of the two

VFTs [9] In a first orientation, lobe-IIs are far apart

in the absence of agonist or in the presence of

antag-onist This orientation is, therefore, called ‘resting’ A

second orientation is observed in the presence of

agon-ist and is therefore considered active In that case,

lobe-IIs are in close contact and one VFT is closed,

whereas the other remains open More recently, a

structure has been solved in the presence of both

agon-ist and Gd3+ [10] In that case, the same active

orien-tation is observed, but both VFTs are in a closed state

(Fig 2A) These data illustrate that the dimer of mGlu

VFTs can have at least three conformations: the

resting-open-open (Roo, resting orientation with both

VFTs in an open state), the asymmetric

active-closed-open (Aco) and the symmetric active-closed-closed

(Acc) conformations

How can agonist binding affect the relative

orienta-tion of the VFTs? Much can be deduced from analysis

of the interface between the subunits at the level of

lobe-II when both VFTs are maintained in the active

orientation This interface revealed major charge

repul-sion if both VFTs are open, consistent with the great

instability of this form of the dimer (note this is

deduced from modelling studies, because this form of

the receptor has never been observed) [10] In contrast,

in the Aco state, the interface consists of a number of

ionic interactions between the two subunits Finally,

when both VFTs are closed (Acc state), four acidic

side chains are facing each other, creating a

cation-binding site that likely needs to be occupied for this

state to be stable [10]

We recently examined whether both Aco and Acc

conformations lead to similar properties of the dimeric

mGlu receptor [31] To that aim, we used the

quality-control system of the GABAB receptor to generate

mGlu receptor dimers composed of two distinct bind-ing sites, either from two distinct mGlu receptors or from a wild-type and a mutated VFT This allowed us

to show that a single ligand per dimer stabilized the Aco conformation, leading to partial activation of the receptor (Fig 3A) Only upon binding of two agonists per dimer was the Acc state reached, leading to full activity [31] Of interest, this fully active state is further stabilized by cations such as Ca2+or Gd3+

Although two glutamates bind in a dimeric mGlu receptor, no strong cooperativity could be measured

by analysing the Hill coefficient However, functional analysis suggests a positive cooperativity between both sites Indeed, agonist potency is 3–5 times lower in a receptor dimer that possesses a single wild-type site Moreover, our data also revealed that when one VFT

A

B

Fig 3 Activation mechanism of homodimeric class C GPCRs and its regulation by allosteric modulators (A) In the absence of agon-ist, the receptor is in a resting state (Roo-HD), and switches to a partially active state upon binding of a first agonist [Aco-HD (*) ], and

to a fully active state upon binding of a second agonist (Acc-HD*) Binding of an inverse agonist in the HD stabilizes the fully inactive ground state of the receptor, whereas binding of a positive allo-steric modulator further stabilizes the fully active state of the agon-ist-bound dimer (B) Schematic representation of the functioning of the class C GPCR after deletion of the large extracellular domain, and illustrating the main three states: the basal state HD that can generate basal activity of the receptor, the ground inactive state HDg stabilized by inverse agonists, and the fully active state stabil-ized by positive allosteric modulators.

is in the closed state, it stabilizes the associated VFT

in the closed state Such observations are in contrast

to the negative allosteric interaction reported between

the mGlu1-binding sites using binding experiments on

purified and soluble VFTs [32] However, this may be

explained by the absence of the other part of the

receptor (the CRD and the HD), as well as by the

absence of cations that stabilize the Acc state

Although two agonists per dimer are required for full

activation of homodimeric class C GPCRs, a single

agonist is sufficient to fully activate the heterodimeric

receptors This has been demonstrated in the case of

the GABAB receptor in which GABA binds in the

GABAB1 VFT only [33] Surprisingly, although the

GABAB2 subunit also possesses a VFT, no natural

ligand probably binds in this domain, as illustrated by

the absence of selective conservation of residues in the

putative binding pocket during evolution Even though

the GABAB2VFT does not bind GABA, it is necessary

for GABAB receptor activation Indeed, among the

various combinations of GABAB1–GABAB2 subunit

chimera generated, only those possessing both the

GABAB1 and GABAB2 VFTs display agonist-induced

activity [34] This is consistent with the proposal that a

change in the relative orientation of the VFTs in the

dimer is associated with receptor activation As shown

for the mGlu receptors, isolated GABAB1and GABAB2

VFTs form dimers (heterodimers in that case), and this

increases affinity for agonists but not for antagonists

[29] This effect likely results from a stabilization of the

closed state of the agonist-bound GABAB1 VFT by

the GABAB2VFT, a proposal that is reminiscent to the

positive allosteric coupling between the VFTs of mGlu

receptors described above Although closure of the

GABAB1 VFT is sufficient to fully activate the

recep-tor, whether the associated GABAB2 VFT also has to

reach a closed empty form remains unknown

As observed in the GABABreceptor heterodimer, a

single agonist is also likely to be sufficient to activate

the sweet and umami taste receptors, the sweeteners

aspartame and neotame interacting in the T1R2 VFT

of the sweet taste T1R2 : T1R3 heterodimer, whereas

glutamate binds in the T1R1 VFT in the umami taste

T1R1 : T1R3 heteromer [35] However, in contrast to

the GABAB2 subunit, the T1R3 VFT-binding site is

very well conserved during evolution, suggesting that

natural ligands bind in this subunit also Such ligand

remains to be identified, but may likely act in synergy

with the oligosaccharides and glutamate

In summary, interaction between VFTs is crucial for

class C GPCR activation Although agonist binding

stabilizes the closed state of the bound VFT, this does

not correspond to the major difference in the resting

and active conformation of the VFT dimer Indeed, whether one or two ligands interact in this dimeric unit, the main consequence is the stabilization of a new relative orientation of the VFTs But how is this transmitted to the HDs within the dimer?

Allosteric coupling between the extracellular and heptahelical domains within the dimer

Whether agonist binding interacting in one VFT of the dimer activates the HD of the same subunit and⁄ or that of the associated subunit has been carefully exam-ined in both heterodimeric GABAB and homodimeric mGlu receptors (Fig 2B)

In the case of the GABAB receptor, it was soon observed that the GABAB1 subunit could not activate the G-protein even when its ER retention signal was mutated [28,36] As such it was soon proposed that the GABAB2subunit was responsible for G-protein activa-tion This was firmly demonstrated in several ways First, mutations into either the i2 or i3 loop of GABAB2 suppressed G-protein activation by the het-erodimer, whereas the equivalent mutation in GABAB1 had a minor effect [37,38] Second, a receptor combi-nation composed of the VFTs of both GABAB1 and GABAB2, but of two HDs from GABAB2, can activate G-proteins upon agonist application, although with a much lower efficacy than the heterodimer, demonstra-ting that the HD of GABAB2 possesses enough of the molecular determinants required for G-protein coup-ling [34] Finally, it has recently been shown that this GABAB2 HD expressed alone can be activated by CGP7930 [17], a positive allosteric modulator of the GABAB receptor It was therefore concluded that trans-activation occurs in the GABABreceptor, GABA binding in the GABAB1 VFT leading to activation of the GABAB2HD

Although GABAB1 VFT binds the agonist and the GABAB2 HD couples to G-protein, a chimeric con-struct composed of these two domains cannot be acti-vated by agonists when expressed alone [34] Normal functioning can be restored when such a chimeric con-struct is coexpressed with the reverse chimera bearing the GABAB2 VFT and the GABAB1 HD, demonstra-ting the importance of dimer formation for function

Of interest, note that in the case of this combination

of chimeric subunits, cis-activation occurs, because the agonist binding domain and the G-protein coupling domain are part of the same subunit

Coupling between ligand binding and HD activation has also been recently examined in the homodimeric mGlu receptors As described above, by manipulating

each subunit in a receptor dimer, it was shown that

the monoliganded dimer of VFTs in the Aco

mation led to partial activity, whereas the Acc

confor-mation with two bound agonists led to a full activity

of the receptor [31] By examining the effect of a point

mutation known to prevent G-protein activation in the

i3 loop of either HD, it was shown that both the Aco

and Acc conformations of the VFT dimer activate

either one or the other HD [31] This demonstrates

that both cis- and trans-activation occur in

homo-dimeric mGlu receptors (Fig 2B)

Taken together, these data highlight the need for

dimer formation for the signal transmission from the

VFT to the HD, and also show that in homodimeric

receptors, the signal from one VFT can be transmitted

to either HD These observations fit nicely with the

proposal that the stabilization of a specific relative

orientation of the VFTs by agonists, also stabilizes a

specific association of the HDs leading to their

activa-tion (Fig 3A) Such a proposal is supported by recent

data obtained using a FRET approach and showing a

specific change in the general conformation of the HD

dimer upon receptor activation [39]

Allosteric functioning of the HD

of class C GPCRs

As observed for class A GPCRs, some class C

recep-tors display constitutive, agonist-independent activity

As described above, because the VFTs have the ability

to close in the absence of agonist, spontaneous closure

may well be at the origin of constitutive activity in

some of these receptors, as observed for the GABAB

receptor [40] Indeed, in that case, competitive

antago-nists act as inverse agoantago-nists by preventing the

sponta-neous closure of GABAB1 VFT However, in the case

of the mGlu1 and mGlu5 receptors, their constitutive

activity was not inhibited by competitive antagonists,

demonstrating that their HD can reach an active state

even when the VFTs stay open This was further

dem-onstrated in two ways First, noncompetitive mGlu1

and mGlu5 antagonists known to bind directly in the

HD of these receptors were found to have inverse

agonist properties [41,42] Second, the HD of mGlu5

expressed alone (mGlu5 receptor deleted of its large

extracellular domain) was found to display the same

constitutive activity as the full-length receptor, an

activity that can be inhibited by inverse agonists

bind-ing in this domain [16]

In addition to the noncompetitive antagonists,

posit-ive allosteric modulators of class C GPCRs also bind

in the HD [43] In most cases, these compounds do

not have agonist activity, but potentiate both the

effic-acy and the potency of agonists However, when the large extracellular domain was deleted from the recep-tor these compounds act as full agonists [16], and are therefore able to stabilize a new fully active conforma-tion of the HD (Fig 3B) As such, as observed with rhodopsin, the HD of class C GPCRs can exist in at least three major states: an HDg (ground) state, which corresponds to the totally inactive state stabilized by inverse agonists; an HD state, which is able to activate G-proteins although with a low efficacy (this state being responsible for the constitutive activity of some receptors); and an HD* state, which corresponds to the active state of the receptor stabilized by positive allosteric modulators (Fig 3B)

Why are the mGlu5 positive modulators unable to activate the full-length receptor although they can fully activate an isolated HD? This indicates that the HD* state cannot be reached if the VFT dimer is not in the active orientation This suggests that the HD* state is likely associated with a specific orientation of the HDs

in the dimer that can be reached when the extracellular parts of the subunits are deleted (Fig 3A)

Taken together, these observations show that the

HD of class C GPCRs can oscillate between various conformational states, each being stabilized either by synthetic ligand directly interacting in this domain, or

by specific conformations of the VFT dimer

Conclusion Although class C GPCRs appeared to be more com-plex proteins than the class A receptors, because of their multiple domains and their association into con-stitutive dimers, much information on their activation process has been gained in recent years These findings illustrate the importance of allosteric transition between various conformations of each domain These transitions can be summarized as follow The extracel-lular binding domains (the VFTs) can oscillate between

an open and a closed conformation, the latter being stabilized by agonists The relative orientation of the VFTs also oscillate between at least two positions, the resting ‘R’ orientation, and the active ‘A’ orientation, the latter being stabilized when at least one VFT is in

a closed conformation, and further stabilized if both VFTs are closed The HDs can also exist in at least three states, the HD state responsible for the constitu-tive activity of some receptors, the fully inacconstitu-tive state HDg stabilized by inverse agonist, and the fully active state HD* stabilized by the active form of the dimer of VFTs (the Acc conformation)

Such complex functioning of these receptors offers

a number of possibilities for allosterically regulating

their activity using compounds acting at various sites

of the receptor One such possibility is to further

sta-bilize the closed state of the VFT after agonist binding

Such a possibility has been proposed for the positive

allosteric effect of Ca2+ on the GABAB receptor [44]

Another possibility is to stabilize the Acc

conforma-tion of the dimer of VFTs, as seen with Gd3+ in the

mGlu receptors [10] As already reported for many

class C GPCRs, compounds directly interacting with

the central pocket of the HD also stabilize a specific

conformation of this domain and affect functioning of

the receptor (acting as inverse agonists or positive

modulators), but other possibilities exist, such as

mole-cules acting at the contact interface between the HDs

Eventually, although the specific role of the CRD in

the activation process is not known, compounds acting

at this level may also influence functioning of the

receptor In support of this idea, large sweet proteins

such as brazzein appear to contact the CRD of the

T1R3 receptor subunit [45] Accordingly, class C

GPCRs represent good targets for drug development

not only because of their important physiological roles,

but also because the large number of possibilities for

regulating their activity

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