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Related Concept Videos

G-protein Coupled Receptors01:21

G-protein Coupled Receptors

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G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
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G Protein-coupled Receptors01:15

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G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
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Activation and Inactivation of G Proteins01:22

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Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
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Transducer Mechanism: G Protein–Coupled Receptors01:30

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G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical,...
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Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
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G-protein-coupled receptor expression and purification.

Karolina Corin1, Lotta T Tegler, Sotirios Koutsopoulos

  • 1Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 21, 2014
PubMed
Summary

This study presents three methods for producing stable G-protein-coupled receptors (GPCRs) using cell-free systems, HEK cells, and E. coli. These protocols facilitate GPCR purification and analysis for better understanding of their structure and function.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • G-protein-coupled receptors (GPCRs) are crucial cell membrane proteins regulating biological functions and serving as drug targets.
  • Limited knowledge of GPCR structure and function hinders research and therapeutic development.
  • Producing sufficient quantities of soluble, functional, and stable GPCRs is a primary bottleneck in GPCR studies.

Purpose of the Study:

  • To present three distinct protocols for the production and solubilization of stable GPCRs.
  • To overcome the limitations in current GPCR production methods.
  • To enable further biophysical and biochemical analysis of GPCRs.

Main Methods:

  • Cell-free in vitro translation systems for GPCR production.
  • Human embryonic kidney (HEK) cell expression system for GPCR production.
  • Escherichia coli (E. coli) expression system for GPCR production.
  • Immunoaffinity chromatography and gel filtration for receptor purification.

Main Results:

  • Successful production and solubilization of stable GPCRs using the described protocols.
  • Purified stable GPCRs amenable to standard biophysical techniques and biochemical assays.
  • Demonstration of versatile approaches for GPCR production across different expression systems.

Conclusions:

  • The presented protocols offer effective strategies for producing stable GPCRs.
  • These methods facilitate the purification and analysis of GPCRs, advancing their study.
  • The findings contribute to overcoming key challenges in GPCR research and drug discovery.