Use of mutants in understanding seedling development.pptx
Analysis gpcr-dimerization
1. Analysis done by :
Léo Bunel (Bordeau University)
Asmae Lguensat (Cadi Ayyad University)
Neuroplasticity course
With Pr.Marc Landry
Drug discovery Today 2008
2. Outlines
• Introduction
• GPCR dimers are ubiquitous
• Functions of GPCR dimers
• Criticism of the ubiquity of dimers
• Strategies to identify physiologically relevant dimers
• Paradigm shift from molecular-based targets to
pathway-based in vivo systems
• Conclusion
3. Introduction
GPCRs :
• mediate the intracellular effects of different extracellular
stimuli : light,odorants, neurotransmitters and hormones
• mediating multitude of physiological and pathophysiological
processes
• primary target for 26.8% of prescription drugs
4. • A single GPCR can :
Activate different G-proteins and associated
effector systems
Activate different intracellular pathways by
interacting with and activating other proteins.
5. Alterations in the levels of
RGS proteins will greatly
influence GPCR-mediated
responses without altering the
levels of active receptor.
Richard R. Neubig & David P. Siderovski,Nature Reviews Drug
Discovery 1, 187-197 (March 2002),doi:10.1038/nrd747
• One of many GPCR interacting proteins
Factors influencing GPCRs activity : RGSs as an example
6. GPCR dimerisation?
• GPCR dimerization (or oligomerization), the
ability of these receptors to interact with each
other in conjunction with agonist
8. GPCR dimers are ubiquitous
• GABA receptors
GABA A/C receptor
GABA B receptor
Ionotropic
Metabotropic
GABABR1 GABABR2
Example : GABA B receptor:
9. GABABR1 GABABR2
Responsible for ligand
binding
required for
G-protein coupling
retained in the ER when
expressed alone
Coexpressed with GABAR1
and targeted in the cell
membrane
functional synergy
10. Functions of GPCR dimers
ER trapped mutant
receptor?
• mutant receptor genes produce a
receptor that is synthesized and
glycosylated in the endoplasmic
reticulum (ER) but does not reach
the cell surface.
early studies exploited this
phenomenon to identify
potential GPCR dimers
Pharmacological chaperones
?
• pharmacologically selective
compounds that stabilize some
conformational mutants, promoting
their proper transport to their site of
action where, in many cases, they
can be functional
(Bernie et al, 2004)
11. • Receptor activity-modifying proteins:
• Non-GPCR chaperons.
• A family of proteines (RAMP1,2,3) that bind to cell surface
receptors and alter their specificity, signaling mechanism, or mode
of intracellular transport.
• have profound effects on receptor pharmacology
stabilize some conformational mutants, promoting their proper
transport to their site of action
12. Criticism of the ubiquity of dimers
• Are all GPCRs born and functionning as dimers?
▫ Evidence through Immunoprecipitation or FRET/BRET
▫ Model overexpressing GPCR: non physiological.
▫ Exemple: NK1R false dimers.
13. Criticism of the ubiquity of dimers
• Are all GPCRs born and functionning as dimers?
▫ Different conditions of dimerization. Rhodopsin
dimers activators of heterotrimeric G-prot.
▫ GPCRs can be functionally monomeric proteins
We need to avoid generalizations. The immense
diversity of GPCRS Structures also applies to their
dimerization.
15. Methodology
• Techniques to higlight dimerization:
Bioluminescence/Fluoresence Resonance energy transfer
DBT: Donor BleedThrough; ABT: Acceptor BleedThrough; ET-SE: Energy-Transfer-Sensitized
Emission; CTZ: coelenterazine (luciferin, luciferase substrate)
http://www.columbia.edu/cu/chemistry/groups/min/BASFI.htm
16. Strategies to identify physiologically
relevant dimers.
• How to deal with GPCR modulation crosstalk?
▫ Investigated receptors both expressed in one same cell
type to be physiologically relevant.
Co-immunoprecipitation
GMO Animals with tagged versions of target receptors can
also be used with RET.
▫ Then the role and possible use as a drug target of the
receptor must be investigated.
17. Paradigm Shift from molecular-based
targets to pathway-based in vivo
systems.
• Evolution of the drug design for GPCRs.
▫ Ligand based design >> new methods to assess GPCR structure >>
pool of known GPCR >> Receptor based design
▫ Ability to developp new drugs surprisingly decreased over the recent
years.
In vitro assays aren’t representative for in vivo efficacy and toxicity of
drugs.
▫ Need for in vivo hight-throughput screening (HTS).
▫ Pharmaceutical industries must focus on a pathway more than on a
simple target molecule.
18. Paradigm Shift from molecular-based
targets to pathway-based in vivo
systems.
• New strategies of screening:
▫ Primary cultures of cells expressing the receptors of interest in vivo . Use for
secondary screening or agonist screening (difficult culture). GPCR dimer
structure and function caracterization.
▫ Existing cell lines that mimic cell systems found endogenously (hCMEC/D3
and melatonine MT1 receptor/GPR50 dimerization, siRNA knock down
confirmation)
▫ Pluripotent embryonnic stem cells (ESC).
Propagate in vitro, differentiate in almost every cell
Still difficult (ex : heterogenicity in derived cells)
▫ ESC other advantage: they can be generated from genetically modified
animals which express a pathology to serve as cellular models of this disease
for HTS.
19. Conclusion
• In spite of the challenges associated with GPCR
dimers, it is clear that they will continue to
attract an inordinate amount of attention
because they are likely to lead to novel insights
into GPCR physiological pathways and
pathophysiology.
A short oral Remider of GPCRs action : they are made of 7 TMD, when they are activated Upon occupancy by the agonist, the receptor conformation is shifted to the active state, causing the heterotrimeric Gprotein to dissociate. it can stimulate or inhibit effector proteins
By their activation ..
The pathophysio processes : Involved in growth and metastasis of some types of tumors by activating p53 gene
The stimulation of two different effector systems for GPCRs like the b2- adrenergic receptor (b2AR) can lead to the concomitant activation of adenylyl cyclase via Gas proteins and the ERK1/2 MAP kinases via the recruitment of b-arrestin.
There is clinical significance to such findings because the b-blocker carvedilol antagonizes the Gas pathway while stimulating the b-arrestin pathway [20]. Such pharmacological and functional pleotropism also serves to increase the repertoire of GPCR-mediated responses
RGS proteins as G GTPase-accelerating proteins (GAPs). When the agonist is bound to the receptor, so we talk about the activated state, gtp is bound to apha subunit, the subunites of gprot dissociate from each other and give a signal, the rgs prot interfere in order to stop and reduce g prot signaling by binding to alpha gtp , in this stage, gtp will be dephosphorilated into gdp and the g prot subunits will reasociate again and come back to the iactive state.
The binding of agonists to G-protein-coupled receptors (GPCRs) enhances the receptor guanine-nucleotide-exchange (GEF) activity, leading to the loading of GTP by the G subunit, conformational changes in the G switch regions (I, II and III), producing the active *-confirmation, dissociation of the G–G complex, and resultant effector interactions (denoted by 'Signal' in the figure). RGS proteins reduce GPCR signalling by accelerating the rate of GTP hydrolysis by the G-protein -subunit, which leads to G–G reassociation. Inhibiting the binding of the RGS-box to GGTP in this case would lead to a prolonged lifetime of the G subunit in the GTP-bound state, enhancing the receptor-stimulated response through increased levels of free GGTP and G subunits
Therse prot are known with a variety of terms including signalsome and signaplex
These complexes are involved in all the aspects of GPCR biology including receptor targeting, pharmacology, potency and signaling specificity.
http://journal.frontiersin.org/article/10.3389/fendo.2011.00002/full
In the absence of any physical or functional interaction, the activation of two different GPCRs induces two independent signaling pathways A and B, which consequently result in two independent effects 1 and 2. (II) Physical interaction of two different GPCRs results in the activation of the heteromer-specific signaling pathway C that will be responsible of a downstream effect 3. (III) In the absence of physical interaction, a functional interaction can be observed when signaling pathways A and B crosstalk to produce effect 4.
There are many examples of gpcr dimers, like the case of taste receptors and rhodopsine receptors, here we will focuse only on the example
GABA B receptor forms heterodimer composed by GABA B1 and GABA B2. GABA B1 is responsible for ligand binding in N-terminal VFT domain, whereas the VFT of GABA B2 fails to bind any known ligand. PAMs bind to GABA B2 transmembrane domain to potentiate the effect of agonist.
http://journal.frontiersin.org/article/10.3389/fphar.2014.00012/full
When expressed alone GABABR1 is retained in the ER because it requires the coexpression of GABABR2 to be properly targeted to the cell surface.
Whereas the GABAB1 subtype binds various GABA Breceptor ligands, it is not functional when expressed alone. Only coexpression with GABA
B2 that is incapable of GABA B receptor ligand binding but promotes efficient cell surface trafficking and G protein coupling, allows the formation of a functional GABAB receptor
We gonna see an example of these chaperons
Gpcrs are considrerd as chaperons
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631373/
A family of proteins that bind to CELL SURFACE RECEPTORS and alter their specificity, signaling mechanism, or mode of intracellular transport.
three single-pass transmembrane proteins
They associate in the endoplasmic reticulum with a GPCR known as calcitonin receptor-like receptor (CLR). The RAMPs and CLR by themselves have poor abilities to reach the cell surface and cannot bind any known endogenous ligand. However, the complexes are translocated to the cell surface where they respond to CGRP, adrenomedullin and adrenomedullin 2 via CGRP, AM1 and AM2 receptors
Evidences have been gathered for some cases such as the familiy C including GABABRs, with techniques like immunoprecipitation and bioluminescence or fluorescence resonance energy transfer.
The main criticism to this techniques is the overexpression of GPCR in transfected cells, while they are expressed at fewer levels in vivo. We can therefore imagine non-physiological interactions.
Exemple: NK1R can interact only at high level of expression, by a non specific receptor interaction
Conditions of dimerization could be different for every proteins: some might just exist transiently, and/or be formed due to agonist binding.
Studies on Rhodopsin receptors suggest its dimeric form serves as a functional signaling of activation for a heterotrimeric G-protein.
Other studies have shown that some GPCRs can be functionally monomeric proteins, and capable of activating G-proteins as monomers (Rho, B2-Adre, Neurotensin recceptors).
First of the previously evoked methods, co immunoprecipitation consists in the binding of one of the subunits by and antibody, itself fixed on a plate. After washing, only remain the target subunit and every protein bound to it. This permits to isolate monomers and heteromers and thus deduce their composition.
This second method is the more subject to criticism. It is based on the transfer of energy from a donor, which is used to tag a first protein, to an acceptor, used to tag the second protein. The acceptor will then emit light used to detect this interaction. This needs a close link between the two tagged proteins, indicating their dimerization. However, if the molecules stand too close to each other the energy transfer can happen without needing a molecular binding. This situation happens in case of excessive overexpression of the proteins of interest.
Make sure the investigated receptors are expressed in at least one same cell type to be physiologically relevant. The standard test to adress this issue is co-immunoprecipitation but it doesn’t differenciate cellular location of the proteins.
GMO Animals models with tagged versions of target receptors can also be used with RET (physiol. Lvl of receptor).
GPCRs targeting drugs used to be designed by chemically modifying their known agonist.
It only required knowledge of the ligand.
With the development of molecular cloning, transfection and overexpression of proteins, and the development of automated assays to screen large chemical libraries, we have a large pool of known GPCRs that could be drug targets.
However the ability to developp new drugs has been decreasing over the recent years. In vitro assays aren’t representative for in vivo efficacy and toxicity of drugs.
There is a need for in vivo hight-throughput screening (HTS) techniques to increase screening efficiency.
Pharmaceutical industries must focus on a pathway more than on a simple target molecule.
Identification of in vivo cells expressing the receptors of interest. Ensure they respond well even in cell culture. The tests would then be led on primary cell cultures (ex pre-eclampsia and AT1-B2 dimers on platelets), for secondary or agonist panel screening (because of the difficulties encountered with their culture).
Exploration of the power of monomeric-GPCR agonist to activate dimers (ex: opioïd agonist 6’GNTI thought to be KOR agonist has a much higher affinity for KOR-DOR dimers), and the impact of allosteric modulation of dimers/monomers. Powerfull tool for GPCR dimer structure and fuction caracterization.
Existing cell lines might also mimic some cell systems found endogenously like hCMEC/D3, a human endothelial cerebral cell line expressing melatonine MT1 receptor and GPR50, an orphan receptor which has been shown to inhibit MT1 by dimerizing with it. This was confirmed by siRNA knock-down of MT1, but could also have been proved by primary cell culture from MT1 KO animals.
The greatest hope for new screening systems relies on pluripotent embryonnic stem cells (ESC). ESC can propagate in vitro and differentiate in almost every cell type, including GPCR-expressing cell types.
The generation of terminally differentiated cells with homogenous phenotype remains difficult (ex : heterogenicity in stem-cell derived dopaminergic neuronal cells)
ESC have another advantage: they can be generated from genetically modified animals which express a pathology to serve as cellular models of this disease fo HTS.
