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Related Concept Videos

G Protein-coupled Receptors01:15

G Protein-coupled Receptors

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...
GPCRs Regulate Adenylyl Cylase Activity01:09

GPCRs Regulate Adenylyl Cylase Activity

Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of cells.
Two...
Transducer Mechanism: G Protein–Coupled Receptors01:30

Transducer Mechanism: G Protein–Coupled Receptors

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, 7TM, or...

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Related Experiment Video

Updated: May 11, 2026

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
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aGPCR-HEK: A Stable High-Expression Inducible Mammalian Cell Expression System for Adhesion GPCR Structural Biology

David M Favara1,2,3, Christopher G Tate1

  • 1Division of Structural Studies, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.

Bio-Protocol
|March 12, 2026
PubMed
Summary

Researchers developed aGPCR-HEK, a novel method for producing correctly folded adhesion G protein-coupled receptors (aGPCRs) in mammalian cells. This system enables milligram-scale purification of aGPCRs for structural studies like cryo-electron microscopy (cryo-EM).

Keywords:
Adhesion GPCR (aGPCR)HEK293S GnTI- TetRInducible expressionLentivirusStable cell lineTetracycline induction

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Area of Science:

  • Structural Biology
  • Biochemistry
  • Cell Biology

Background:

  • Adhesion G protein-coupled receptors (aGPCRs), including ADGRL4, are implicated in various cancers.
  • Conventional insect cell expression systems often fail to produce sufficient correctly folded aGPCRs for structural analysis.
  • Cryo-electron microscopy (cryo-EM) requires high-purity, well-folded protein samples.

Purpose of the Study:

  • To establish a robust mammalian cell-based expression system for producing milligram quantities of correctly folded aGPCRs.
  • To enable structural and biochemical studies of aGPCRs, particularly ADGRL4.
  • To overcome limitations of existing expression systems for aGPCRs.

Main Methods:

  • Development of a stable, tetracycline-inducible HEK293S GnTI- TetR cell line.
  • Lentiviral transduction for efficient aGPCR gene integration.
  • Flow cytometry enrichment of cells with leaky GFP expression to enhance inducibility.
  • Adaptation to suspension culture for large-scale protein production.
  • Downstream purification and cryo-EM analysis.

Main Results:

  • The aGPCR-HEK system reproducibly yields milligram-scale quantities of folded aGPCRs.
  • The protocol facilitates the production of structural biology-grade protein.
  • Successfully produced N-terminally HA- and GFP-tagged aGPCRs suitable for purification.

Conclusions:

  • The aGPCR-HEK system provides a reliable method for expressing and purifying aGPCRs for structural and biochemical investigations.
  • This mammalian expression system overcomes previous challenges in obtaining sufficient folded aGPCR protein.
  • The protocol is adaptable for both adherent and suspension cell cultures, enhancing its versatility.