GPCR Oligomerization Overview
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What is the primary purpose of Co-Immunoprecipitation (Co-IP)?

  • To capture proteins bound to a specific target protein. (correct)
  • To perform Western blotting on isolated proteins.
  • To analyze the molecular weight of proteins.
  • To determine the structure of proteins.

    • Fluorescence Resonance Energy Transfer (FRET) relies on which of the following phenomena?

  • Distance-dependent energy transfer between fluorescent donor and acceptor molecules. (correct)
  • Energy absorption by fluorescent receptors.
  • Chemical reactions between fluorescent molecules.
  • Direct binding of fluorescent proteins to one another.

    • What significant structure was revealed through MALDI mass spectrometry of GPCR and G protein complexes?

  • A trimer consisting of three identical GPCRs.
  • A dimer formed from two G proteins.
  • A monomer of a GPCR.
  • A pentameric unit of two GPCR monomers and one heterotrimeric G protein. (correct)

    • Which technique would most likely be used to analyze receptor complexes after crosslinking proteins?

    <p>Western blot analysis to detect higher molecular weight forms.</p> Signup and view all the answers

    What does the analysis of protein complexes through co-immunoprecipitation help identify?

    <p>New binding partners and their binding affinities.</p> Signup and view all the answers

    What characteristic of bivalent ligands allows them to potentially increase receptor heterodimerization?

    <p>They can bind to two different receptors simultaneously.</p> Signup and view all the answers

    What is the main advantage of using dimeric ligands over monovalent ligands?

    <p>Dimeric ligands can induce dimerization of receptors.</p> Signup and view all the answers

    What is a specific characteristic of the heterodimer-specific ligand?

    <p>It binds to a conformation present only in heterodimers.</p> Signup and view all the answers

    Which statement best describes the potential of bivalent ligands in drug development?

    <p>They may provide increased affinity and selectivity.</p> Signup and view all the answers

    How do dimeric ligands operate to enhance receptor interactions?

    <p>By crosslinking two receptors through an appropriate linker.</p> Signup and view all the answers

    What is the minimal oligomeric arrangement of GPCRs?

    <p>Dimer</p> Signup and view all the answers

    How does heterodimerization impact GPCRs?

    <p>It enhances their ability to respond to ligands</p> Signup and view all the answers

    What is one role of GPCR dimerization during receptor maturation?

    <p>It facilitates transport from the ER to the cell surface</p> Signup and view all the answers

    What are receptor heterodimers expected to exhibit compared to their individual protomers?

    <p>Distinct signaling and trafficking properties</p> Signup and view all the answers

    What effect can ligand binding have on GPCR dimers?

    <p>It can either promote or inhibit dimer formation</p> Signup and view all the answers

    What is an assumption about GPCRs that has been altered by recent evidence?

    <p>They are capable of forming oligomeric complexes</p> Signup and view all the answers

    What does it mean for GPCR heterodimers to bind heterodimer-specific ligands?

    <p>Ligands can only bind when in a heterodimeric complex</p> Signup and view all the answers

    What is the primary function of GPCR oligomerization in terms of cell biology?

    <p>To reshape our understanding of receptor functions</p> Signup and view all the answers

    Which example illustrates ligand-promoted receptor hetero-oligomerization?

    <p>Dopamine receptor (D2R) and somatostatin receptor (SSTR5) hetero-oligomers</p> Signup and view all the answers

    What characterizes constitutive dimerization processes in GPCRs?

    <p>They may occur independently of ligand interactions.</p> Signup and view all the answers

    How does GPCR heterodimerization affect signaling pathways?

    <p>It leads to potentiation and attenuation of signaling.</p> Signup and view all the answers

    Which statement about G-protein interactions with GPCR dimers is generally true?

    <p>A single G-protein interacts with each GPCR dimer.</p> Signup and view all the answers

    What impact does heterodimerization have on receptors?

    <p>It modifies biochemical, pharmacological, and functional properties.</p> Signup and view all the answers

    What is a consequence of the co-internalization of GPCR heterodimers?

    <p>It allows the internalization of one receptor upon stimulation of the other.</p> Signup and view all the answers

    What is an example of positive ligand binding cooperativity in GPCRs?

    <p>δ- and κ-opioid receptor heterodimer</p> Signup and view all the answers

    Which of the following techniques is used for assessing GPCR internalization?

    <p>β-arrestin recruitment assay</p> Signup and view all the answers

    Which model describes the interaction of GPCRs in heterodimerization?

    <p>The contact dimer or lateral packing model</p> Signup and view all the answers

    Which cell lines are commonly used in recombinant systems to study GPCRs?

    <p>CHO and HEK293</p> Signup and view all the answers

    What can inhibit the internalization of a GPCR heterodimer?

    <p>Presence of a protomer resistant to endocytosis</p> Signup and view all the answers

    What type of ligands can be used to selectively target GPCRs?

    <p>Synthetic agonists and biased agonists</p> Signup and view all the answers

    To study drug-receptor interactions and signal transduction, what method is commonly used?

    <p>Recombinant cell lines overexpressing a single GPCR type</p> Signup and view all the answers

    What is assessed through the measurement of intracellular calcium in GPCR studies?

    <p>Downstream effectors and second messengers</p> Signup and view all the answers

    Which approach involves tagging GPCRs and β-arrestin for studies?

    <p>BRET assay</p> Signup and view all the answers

    What role do biased agonists serve in GPCR signaling?

    <p>They promote arrestin-dependent signaling pathways.</p> Signup and view all the answers

    What is the approximate maximum range for Resonance Energy Transfer to occur?

    <p>10 nanometers</p> Signup and view all the answers

    Which factor is crucial for efficient energy transfer in FRET?

    <p>Spectral overlap between donor emission and acceptor absorption</p> Signup and view all the answers

    Which of the following statements describes a scenario that results in FRET?

    <p>Conformational change upon binding leads to energy transfer</p> Signup and view all the answers

    Which characteristic defines Bioluminescence Resonance Energy Transfer (BRET)?

    <p>It uses a bioluminescent donor and a fluorescent acceptor.</p> Signup and view all the answers

    What is the emission wavelength of light produced by Rluc in BRET?

    <p>395 nm</p> Signup and view all the answers

    What happens to the mutant GFP2 when it is close to the emitted blue light in a BRET system?

    <p>It emits light at 510 nm after being excited.</p> Signup and view all the answers

    Which process effectively causes 'No FRET' to occur?

    <p>Proteolytic cleavage</p> Signup and view all the answers

    What compound is catalyzed by Rluc in the presence of oxygen during BRET?

    <p>DeepBlueC</p> Signup and view all the answers

    Study Notes

    GPCR Oligomerization

    • G-proteins are the third-largest gene family.
    • Previous assumptions: GPCRs function as monomers.
    • Recent evidence shows they function as oligomeric complexes, primarily dimers.
    • Oligomers are molecular complexes made of several monomeric units.
    • Dimers represent the simplest oligomeric arrangement, these can be homodimers or heterodimers.
    • Receptor heterodimers are a new area of research, changing our understanding of cell biology and drug discovery.
    • Heterodimers act as distinct functional units, differing in binding, signaling, trafficking, and subcellular localization compared to their protomer counterparts.
    • Heterodimerization allows for flexibility in receptor responses to ligands.
    • Heterodimers can bind heterodimer-specific ligands, which bind only when the receptor is in heterodimeric form.
    • GPCR dimerization during its lifecycle, plays a role in ligand-regulated regulation, signal transduction, and pharmacological diversity.
    • Dimerization during maturation and transport to the plasma membrane is essential for correct folding and exit from the ER.
    • Ligand binding can either promote or inhibit dimer formation.
    • GPCR heterodimers can affect both the intensity and selectivity of G-coupled signaling.
    • A single heterotrimeric G protein typically interacts with each GPCR dimer.
    • Heterodimerization can often promote the co-internalization of GPCRs after stimulation of only one protomer.
    • Different models exist to explain how GPCR dimers and oligomers form, such as lateral packing (contact dimer) which involve direct contact between transmembrane domains.
    • Domain-swapped model requires a change in the monomer's integrity by swapping transmembrane domains.

    Significance

    • Heterodimers exhibit distinct functional properties.
    • Increased plasticity in responses to ligands.
    • Heterodimer-specific ligands bind only in the heterodimeric state.
    • Dimerization can affect GPCR maturation and transport to the cell surface.

    Methods of Studying GPCR Oligomerization

    • Crosslinking of proteins (followed by Western blotting) detects receptor complexes.
    • Co-immunoprecipitation (Co-IP) of differentially epitope-tagged receptors followed by Western blot analysis indicates receptor-receptor interactions.
    • Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) assess receptor-receptor proximity in living cells.
    • Mass spectrometry, for example MALDI, identifies reconstituted receptors and their interactions with heterotrimeric G proteins.

    Methods of Detection

    • Co-immunoprecipitation is a useful technique for studying protein-protein interactions.

    FRET - Fluorescence Resonance Energy Transfer

    • FRET is based on efficient distance-dependent energy transfer from the excited fluorescent donor molecule to the acceptor molecule.
    • CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) are commonly used as donor and acceptor pairs in FRET assays.
    • FRET occurs only when the donor and acceptor molecules are within a specific distance range (commonly 4-10 nm).

    BRET - Bioluminescence Resonance Energy Transfer

    • The method used for detection relies on energy transfer between a bioluminescent donor and a fluorescent acceptor.
    • Luciferase (Rluc) from the sea pansy ( Renilla reinformis) is a frequent donor.

    Summary

    • Heterodimers are present in cells and tissues.
    • Heterodimerization changes the biochemical, pharmacological, and functional properties of receptors.

    GPCR Signaling and Coupling Assessment

    • GPCR signaling in cells expressing the receptors can be studied.

    • Recombinant cell lines expressing a single GPCR type can be used to study drug-receptor interactions and signal transduction.

    • Recombinant systems overexpressing multiple receptors and tagging them can assess receptor trafficking and heterodimerization.

    • Host cell lines, like CHO and HEK293, are frequently used in recombinant systems.

    • Functional GPCR assessment includes radioligand binding (for drug interactions), G-protein coupling assessment (e.g., Pertussis toxin, cholera toxin), intracellular calcium measurement (e.g., IP3, cAMP assay), and kinase activity assays.

    Assessing Dimerization and Internalization

    • Dimerization can be assessed using FRET, BRET, etc.
    • Internalization can be measured using β-arrestin recruitment assays.

    Approaches to Target GPCR Heterodimers

    • Bivalent ligands can bind two different receptors simultaneously and induce heterodimerization.
    • Dimeric ligands generated from monovalent ligands might induce and/or enhance dimerization.
    • Heterodimer-specific ligands bind to receptor binding sites only when the receptors form a heterodimer.

    GPCRs As Drug Targets

    • GPCRs are targets for selective synthetic agonists and antagonists.
    • Biased agonists selectively activate arrestin-dependent signaling pathways, independent of G-protein activation.
    • Bivalent ligands target specific GPCR heterodimers.
    • Allosteric modulators can also be used to modify GPCR activity.

    Receptor Tyrosine Kinases (RTKs)

    • RTKs are a type of receptor that plays an important role in cell signaling.

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    Related Documents

    GPCR Oligomerization Lecture 9 - Bioc 325 PDF

    Description

    Explore the fascinating world of G-protein-coupled receptor (GPCR) oligomerization, highlighting how recent findings challenge the traditional view of GPCRs as monomers. Dive into the significance of dimers and heterodimers in understanding cell biology and drug discovery, and learn about their unique properties and functions.

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