GPCR Oligomerization Lecture 9 - Bioc 325 PDF

GPCRs Oligomerization 1 1. Introduce the concept of GPCR oligomerization. 1. Roles in altering the signaling and trafficking properties of individual receptors. 1. Techniques used to study GPCR oligomerization  G-Proteins constitute the 3rd largest family o...

GPCRs Oligomerization 1 1. Introduce the concept of GPCR oligomerization. 1. Roles in altering the signaling and trafficking properties of individual receptors. 1. Techniques used to study GPCR oligomerization  G-Proteins constitute the 3rd largest family of genes  Previous assumptions: GPCRs exist and function as monomeric entities  Recent evidence- function as oligomeric complexes-dimers  Oligomers: molecular complexes that consist of few monomeric units  Dimer (minimal oligomeric arrangement): Homo and Hetero  Receptor heterodimers comprise a new area of research that is reshaping our thinking about cell biology, and drug discovery 3  Heterodimers as distinct functional units: ◦ Receptor heterodimers exhibit distinct binding, signaling, trafficking, and subcellular localization as compared to their cognate protomers  Heterodimerization allows for a large degree of plasticity in the receptors’ ability to respond to ligands ◦ Additional feature: Heterodimer can bind to heterodimer-specific ligands that only bind to the receptors when in a heterodimeric complex 4 Handbook of cell signaling, 2nd edition Sonia Terrillon, Michel Bouvier EMBO Rep. 2004 January; 5(1): 30–34 5  In some cases, dimerization has been shown to have a primary role in receptor maturation and allows the correct transport of GPCRs from the endoplasmic reticulum (ER) to the cell surface  Only correctly folded receptors are allowed to exit the ER 6  Once at the plasma membrane, dimers might become the target for dynamic regulation by ligand binding  Several studies suggest that ligand binding can regulate the dimer by either promoting or inhibiting its formation. ◦ Ex of ligand-promoted formation: dopamine receptor (D2R) /somatostatin receptor (SSTR5) hetero-oligomers  Other studies consider dimerization as constitutive processes that are not modulated by ligand binding ◦ Ex: Ligand-independent CXCR2 dimerization Note: CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth- 7 related gene product beta) to induce cell chemotaxis  It has been proposed that GPCR heterodimerization leads to both positive (+) and negative (−) ligand binding cooperativity  Example of positive cooperativity: δ- and κ-opioid receptors heterodimer 8  It has been proposed that GPCR heterodimerization leads to potentiation (+)/attenuation (−) of signaling or changes in G- protein selectivity.  Example on G-protein selectivity: a loss of Gi coupling has been reported following co- expression of μ- and δ-opioid receptors 9  Most cases: A single heterotrimeric G protein seems to be interacting with each GPCR dimer  However, additional studies should be conducted to determine if this could be generalized for all functional heterodimers 10  Heterodimerization can promote the co-internalization of two receptors after the stimulation of only one protomer. ◦ Ex: B1R/B2R heterodimer  Alternatively, the presence of a protomer that is resistant to agonist-promoted endocytosis, within a heterodimer, can inhibit the internalization of the complex 11 1. Lateral Packing Model (Contact Dimer) 2. Domain-swapped Model 12 In the contact dimer or lateral packing model, GPCRs directly contact each other via exterior residues of the transmembrane domains (monomeric structure remains intact). These dimers may associate at other interacting surfaces leading to the formation of receptor oligomers. Nature Reviews Drug Discovery 1, 808-820 (October 2002) 13 The domain-swapping model requires changes in the integrity of the monomers by swapping of independently folded transmembrane domains between GPCR monomers. Nature Reviews Drug Discovery 1, 808-820 (October 2002) 14 Searching for the Functional Dimer Fingerprint 15  B1 and B2 Receptor Transfection and Internalization Upon Ligand Stimulation NS Des-Arg-BK  B1R and B2R: Bradykinin (100pM) Receptors type 1 and 2 respectively  Bradykinin (BK): agonist for B2R  Des-Arg-BK: specific agonist for B1R B1R-YFP B1R-YFP  When expressed alone in HEK293 cells: ◦ B1R: undergoes des-Arg-BK –induced internalization ◦ B2R: does not internalize after stimulation with des- B2R-YFP B2R-YFP Arg-BK 16  B1R-B2R Heterodimers Co-internalize Upon Ligand Stimulation  When HA-Tagged B1R and YFP-conjugated B2R are co-expressed in HEK293 cells: ◦ B2R: undergoes internalization after stimulation with des-Arg-BK only in the presence of HA-Tagged B1R.(C) ◦ Positive control: B2R-YFP undergoes internalization after the binding of its ligand BK (B) (this usually occurs even in the absence of B1R) A B C Confocal images of B2R-YFP in HEK293 cells co- expressing HA-Tagged B1R and YFP-tagged B2R 17 Heterodimerization of the GABAB Receptor Produces a Functional Receptor NATURE REVIEWS MOL CELL BIOL 3:; 2002 639-650 18  Shift to Gq Coupling in the Dopamine1-Dopamine2 Receptor Heterodimer D1-R D2-R Gs Gi Adenylate Adenylate cyclase cyclase D1-R D2-R DAG Gq Synaptic Plasticity PKC CaMKII Ca2+ IP3 Rashid et al. (2007) PNAS 104, 654-9 19  Heterodimerization leads to a change in the binding pocket that could be specifically targeted to develop heterodimer selective drugs  Development of ‘bivalent’ ligands that specifically target the heterodimer. They might have increased affinity and selectivity over classic receptor ligands.  Development of drug-like molecules that specifically target the heterodimer 20 Approaches to Target Heterodimers 1- Bivalent Ligands A bivalent peptide with two distinct receptor-recognition sites might bind two different receptors simultaneously and induce receptor heterodimerization. If the two receptors pre-exist in an oligomeric state, the ligand might enhance the heteromeric conformation by crosslinking two receptors. NATURE REVIEWS | DRUG DISCOVERY 1: 2002, 808-820 21  Approaches to Target Heterodimers 2- Dimeric Ligands Dimeric ligands generated from two monovalent ligands. Similar to a bivalent ligand, a dimeric ligand that has an appropriate linker might induce or enhance dimerization of two receptors. It is plausible that homodimeric and heterodimeric ligands might selectively bind homomeric and heteromeric receptors, respectively. NATURE REVIEWS | DRUG DISCOVERY 1: 2002, 808-820 22  Approaches to Target Heterodimers Heterodimer specific ligand, which binds to a receptor binding- site conformation that is present only when the receptors are heterodimerized.  Crosslinking of proteins using a variety of agents, followed by Western blot analysis (to detect receptor complexes as a higher molecular weight form)  Co-Immunoprecipitation (Co-IP) of differentially epitope-tagged receptors followed by Western blot analysis (to indicate an interaction between the two tagged receptors)  Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) (as proximity-based biophysical methods that examine receptor–receptor associations under physiological conditions in living cells)  MALDI mass spectrometry of reconstituted receptors with G proteins (revealed the presence of a pentameric unit consisting of two GPCR monomers and one heterotrimeric G protein) 24  Co-immunoprecipitation (Co-IP) is a popular technique to identify physiologically relevant protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.  These protein complexes can then be analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein http://www.piercenet.com/method/co-immunoprecipitation-co-ip 25  Collect the Pellet Discard the Supernatant Images obtained and modified for educational purposes from: http://www.piercenet.com/me thod/co-immunoprecipitation- co-ip http://www.activemotif.com/ images/products/coip_flowch 26 art_big.jpg FRET is based on efficient distance-dependent Resonance Energy Transfer from an excited fluorescent donor to an acceptor fluorescent molecule. 4-10 nm CFP YFP FRET The range over which the Resonance Energy Transfer can take place is limited to approximately 10 nanometers 27  Spectral overlap 4-10 nm  FRET Efficient transfer of energy requires a significant spectral overlap between the donor emission and the acceptor absorption spectra 28  Concept of FRET No FRET FRET 29 Images were captured from the video posted on this link: http://www.youtube.com/watch?v=pMH8zcWa7WA  ZEISS Microscopy Online Campus a) Substarte-sensory protein domain binding FRET b) Ligand-receptor binding conformational change FRET c) Two fluorochromes are attached to each end of a complex unit composed of a sensory domain linked to its binding substrate. Upon stimulation binding conformational change FRET d) FRET Proteolytic cleavage No FRET 30  Bioluminescence Resonance Energy Transfer (BRET)  Based on efficient Resonance Energy Transfer between a bioluminescent donor and a fluorescent acceptor.  BRET uses Luciferase (Rluc) as the donor isolated from the sea pansy Renilla reinformis. BRET2:  In the presence of oxygen, Rluc catalyzes the transformation of its substrate: (DeepBlueC (DBC): coelenterazine derivative) into coelenteramide with concomitant light emission peaking at 395 nm (blue light).  If acceptor mutant GFP2 is within close proximity, it gets excited with the blue light energy and next emits green light at 510 nm.  Energy transfer efficiency between Rluc and GFP is determined ratiometerically by dividing acceptor emission (GFP)/donor emission (Rluc) 31  Bioluminescence Resonance Energy Transfer (BRET2) “BRET occurs when part of the energy from DeepBlueC (DBC)-bound Rluc is transferred to GFP2, which in turn, emits green light. If Rluc and GFP2 are not in close proximity, energy is not efficiently transferred and only the blue light emitted by the Rluc/DBC reaction is detected. GFP2 Excitation Rluc 510nm of GFP2 When Rluc and GFP2 are brought into close proximity by means of a specific biological interaction (Protein 1 and O2 Protein 2 fused to Rluc and GFP2, BRET respectively), energy is efficiently transferred from DBC-bound Rluc to + Substrate: Signal Blue GFP2 resulting in the production of green DBC Light light from GFP2. 395nm Metabolism The BRET2 signal is measured by dividing the amount of green light by the amount of Substrate of blue light. “ 32  Summary Heterodimers exist in natural cells and tissues Heterodimerization modifies Biochemical Pharmacological Functional properties of receptors 33 Classical Methods to Assess Signaling and Coupling of GPCRs 34 Three main routes for in-vitro studies:  Study GPCR signaling in cells endogenously expressing the receptors  Recombinant cell lines overexpressing a single GPCR type to study both drug-receptor interactions and signal transduction (primary screening of GPCRs)  Recombinant Systems overexpressing two or more receptors and tagging of receptors to assess receptor trafficking, signaling, and heterodimerization properties  Examples of host cell lines for recombinant systems: CHO, and HEK293 cell-lines…… 35  Assessing functionality of GPCRs; example: ◦ Radioligand binding for assessment of drug-receptor interaction ◦ Evaluation of type of G-protein coupling: Pertussis toxin, cholera toxin , or study of downstream effectors and 2nd messengers:  Intracellular Calcium Measurement  Measurement of IP3  AC activity or cAMP assay  Activity of kinases (PKA, PKC)…… 36 Assesing GPCR dimerization example: ◦ FRET, BRET,… Assessing GPCR internalization example: ◦ β-arrestin recruitment assay 37 BRET assay. The GPCR is tagged with a RLuc, and β-arrestin is tagged with GFP, or vice versa. 38 Acta Pharmacologica Sinica (2012) 33: 372–384 (a) GPCRs can be targeted using selective synthetic agonists (b) antagonists can be used to block the binding of an endogenous agonist thus preventing receptor activation. (c) Biased agonists are being developed that specifically promote the activation of arrestin-dependent signalling pathways independent of G-protein signalling. (d) bivalent ligands targeting specific GPCR heterodimers. (e) GPCRs can also be targeted using allosteric modulators 39 Receptor Tyrosine Kinases (RTKs)