LOGO Disorders Associated with GPRs Members: 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——VFT VFT ： Venus Flytrap (bilobate 双叶形 )  Can be regulared by allosteric modulator （变构调节剂）  Dimer ——homo or heter

The structural  Four parts 1.VFT(Venus Flytrap): the agonist binding site 2.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 GABA B receptor  Absent disulfide bridge no covalent( 共价的 ) linkage  May have interaction between intracellular tail  ER retention signal (GABA B1 )

Activation mechanism of class-C GPCRs interaction between the VFTs

LOGO 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(Ca 2+ )

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 GABA B receptor in which GABA binds in the GABA B1 VFT only On GABA B receptor

But  GABA B2 is necessary for GABA B  only those possessing both the GABA B1 and GABA B2 VFTs display agonist-induced activity  Why Unknown

Allosteric coupling between the extracellular and HD within the dimer

On GABA B receptor HD of GABA B2 is a important part  Experiment I  Mutations into either the i2 or i3 loop of GABA B2 suppressed G-protein activation.  The equivalent mutation in GABA B1 had a minor effect

 Experiment II GABA B1 VFT GABA B2 VFT GABA B2 HD Demonstrating that the HD of GABA B2 possesses enough of the molecular determinants required for G-protein coupling

 GABA B2 HD expressed alone can be activated by CGP7930, a positive allosteric modulator of the GABA B receptor.  So.. trans-activation occurs in the GABA B receptor  Experiment III

Another Experiment GABA B1 VFT +GABA B2 HD =can not be activated (GABA B1 VFT + GABA B2 HD) +(GABA B2 VFT + GABA B1 HD) = can be activated In this combination subunits cis-activation occurs GABA B1 VFT GABA B2 HD GABA B2 VFT GABA B1 HD

 In a word, dimer conformation takes a very important part in GABA B 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

 Difference Negative ——directly Positive——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  Ro : 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