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Related Concept Videos

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...
GPCR Desensitization01:12

GPCR Desensitization

G protein-coupled receptor (GPCR) signaling plays a crucial role in cell functioning. GPCR desensitization is an equally essential process. It allows cells to respond to changing environments and regain sensitivity to new stimuli while preventing unnecessary stimulation when no longer needed. Prolonged exposure to stimuli leads to GPCR desensitization. It involves blocking the receptors from binding and activating additional G proteins. This inhibits activation of downstream effectors, thereby...
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...
Regulation of Expression at Multiple Steps01:23

Regulation of Expression at Multiple Steps

The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the addition of a...
Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...

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Related Experiment Video

Updated: Jun 3, 2026

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

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Evolution of three human GPCRs for higher expression and stability.

Igor Dodevski1, Andreas Plückthun

  • 1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Journal of Molecular Biology
|March 8, 2011
PubMed
Summary

We developed a method to improve the expression and stability of G-protein-coupled receptors (GPCRs) in E. coli. This technique rapidly evolves functional GPCR mutants, overcoming previous limitations in protein studies.

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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Integral membrane proteins, such as G-protein-coupled receptors (GPCRs), are crucial drug targets but challenging to study due to low expression and stability.
  • Previous methods for directed evolution of membrane proteins in Escherichia coli have limitations.

Purpose of the Study:

  • To enhance the expression and stability of human G-protein-coupled receptors (GPCRs) using directed evolution in E. coli.
  • To develop and validate new screening methods for improved receptor stability and functional expression.

Main Methods:

  • Directed evolution involving cycles of mutagenesis and fluorescence-activated cell sorting (FACS) for GPCRs.
  • Development of a 96-well assay for rapid screening of thermal stability in detergent-solubilized receptors.
  • Application of combined methods to challenging GPCR targets like the tachykinin receptor NK(1).

Main Results:

  • Achieved a 10-fold increase in functional expression for neurotensin receptor 1, retaining wild-type properties and showing enhanced stability.
  • Successfully increased functional expression for three additional human GPCRs.
  • Evolved stable, functionally expressed tachykinin receptor NK(1) mutants at 1 mg/l levels in E. coli, overcoming significant expression and solubilization challenges.

Conclusions:

  • The developed directed evolution and screening methods are highly effective and generally applicable to a wide range of GPCRs.
  • These advancements enable the production of sufficient quantities of stable, correctly folded GPCRs, removing roadblocks for structural and biophysical studies.
  • Cumulative small sequence modifications drive significant improvements in GPCR function and stability.