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Disorders Associated with GPRsMembers:王建博、秦涛、李中印、王宇

The structural and signal transmit mechanism of the class-

C G-protein-coupled receptors

First

What is this? One of the five classes GPCRs

How does the GPCRs classified? Based on sequence similarity. class-A:Rhodopsin( 视紫红质 )-like receptors class-B:secretin( 分泌素 )-like receptors class-C:mGlu-like receptors

What are class-C GPCRs include? neurotransmitters receptors glutamate( 谷氨酸盐 ) receptors GABA receptors the calcium-sensing receptor sweet taste receptors pheromone( 信息素 )receptors

Difference

A large extracellular domain——VFTVFT ： Venus Flytrap (bilobate 双叶形 )

Can be regulared by allosteric modulator （变构调节剂）

Dimer ——homo or heter

The structural

Four parts1.VFT(Venus Flytrap): the agonist

binding site2.CRD(cysteine-rich domain)3.HD(heptahelical domain) C-term

VFT

Tow parts:Lobe-I&Lobe-II

Two conformations ： Open(inactive) & Closed(active)

antagonist & agonist ( 抑制剂 ) ( 激动剂 )

Lobe-I

Lobe-II

CRD

The structure and function are unknown

absent CRD:GABA receptor

HD （ Heptahelical domain ）

Heptahelical

long C-terminal tail

Activity site positive & negative allosteric modulators

independency

How is the signal transduced from one domain to the other?

Homodimeric receptors mGlu receptor

A disulfide-linked dimer Cys-residues

Class-C GPCRs are constitutive dimers

Lobe-I

Lobe-II

Heterodimeric receptors GABAB receptor

Absent disulfide bridge no covalent( 共价的 ) linkage

May have interaction between intracellular tail

ER retention signal (GABAB1)

Activation mechanism of class-C GPCRs

interaction between the VFTs

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On mGlus receptor

(Roo, resting-open-open) (Aco, active-closed-open) (Acc, active-closed-closed)

Are both Aco & Acc conformations lead to similar properties?

Mutated mGlu VFT composed of two distinct binding sites

Roo, none activity Aco, half activity Acc, full activity(Ca2+)

Why?Roo state: This interface revealed major charge

repulsion

Aco state: the interface consists of a number of

ionic interactions

Acc state: four acidic side chains are facing each

other, creating a cationbinding site

Only have two conformations A signal agonist can fully activate a

receptor

Surprisingly GABAB receptor in which GABA binds

in the GABAB1 VFT only

On GABAB receptor

But

GABAB2 is necessary for GABAB

only those possessing both the GABAB1 and GABAB2 VFTs display agonist-induced activity

Why Unknown

Allosteric coupling between the extracellular and HD within the

dimer

On GABAB receptor

HD of GABAB2 is a important part

Experiment IMutations into either the i2 or i3 loop

of GABAB2 suppressed G-protein activation.

The equivalent mutation in GABAB1 had a minor effect

Experiment II

GABAB1

VFTGABAB2

VFT

GABAB2 HD GABAB2 HD

Demonstrating that the HD of GABAB2 possesses enough of the molecular determinants required for G-protein coupling

GABAB2 HD expressed alone can be activated by CGP7930, a positive allosteric modulator of the GABAB receptor.

So.. trans-activation occurs in the GABAB

receptor

Experiment III

Another Experiment

GABAB1 VFT +GABAB2 HD =can not be activated

(GABAB1 VFT + GABAB2 HD) +(GABAB2 VFT + GABAB1 HD)

= can be activated

In this combination subunits cis-activation occurs

GABAB1

VFT

GABAB2 HD

GABAB2

VFT

GABAB1 HD

In a word, dimer conformation takes a very important part in GABAB receptors

On mGlu receptor

Both cis- and trans-activation occur in mGlu receptors

Because the homodimeric structure

Allosteric functioning of the HD of class C GPCRs

HD can exist in three states

HDg states：totally inactive state

HD states：Low active efficiency state

HD* states：High active efficiency state

HD can be regulared by positive & negative allosteric modulators ( 变构调节剂 )

Allosteric modulators are compounds able to regulate

the activity of a receptor by binding at a site distinct from that where endogenous ligands bind

Classification negative allosteric modulators inhibit constitutive activity of the receptor

positive allosteric modulators activate the receptor , can enhance either the

potency （力量） or the efficacy （效力） , or both

DifferenceNegative ——directlyPositive——indirectly(with agonist)

SO…

the action of negative allosteric modulators is less dependent on the concentration of endogenous ligand(agonist)

But positive allosteric modulators is highly dependent on agonist

Experiment

Bay 7620:negative allosteric modulators Ro01-6128: positive allosteric modulators

EC50 :value of glutamate

Why we intrested in allosteric modulators?

Both positive & negative modulators are highly receptor subtype selective.

less side effect , long effect Hydrophobic, allowing them to cross the

blood brain barrier more easily