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Hormones—or any molecule that binds to a receptor, known as a ligand—that are lipid-insoluble (water-soluble) are not able to diffuse across the cell membrane. In order to be able to affect a cell without entering it, these hormones bind to receptors on the cell membrane. When a first messenger, a hormone, binds to a receptor, a signal cascade is set off, causing second messengers, proteins inside the cell, to become activated, resulting in downstream effects.
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Genomics is the science of genomes: it is the study of all the genetic material of an organism. In humans, the genome consists of information carried in 23 pairs of chromosomes in the nucleus, as well as mitochondrial DNA. In genomics, both coding and non-coding DNA is sequenced and analyzed. Genomics allows a better understanding of all living things, their evolution, and their diversity. It has a myriad of uses: for example, to build phylogenetic trees, to improve productivity and...
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Hydronium and hydroxide ions are present both in pure water and in all aqueous solutions, and their concentrations are inversely proportional as determined by the ion product of water (Kw). The concentrations of these ions in a solution are often critical determinants of the solution’s properties and the chemical behaviors of its other solutes. Two different solutions can differ in their hydronium or hydroxide ion concentrations by a million, billion, or even trillion times. A common means of...
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Most plants use the C3 pathway for carbon fixation. However, some plants, such as sugar cane, corn, and cacti that grow in hot conditions, use alternative pathways to fix carbon and conserve energy loss due to photorespiration. Photorespiration is the process that occurs when the oxygen concentration is high. Under such conditions, the rubisco enzyme in the Calvin cycle binds O2 instead of CO2, which halts photosynthesis and consumes energy.
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Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
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Genome-scale identification of cellular pathways required for cell surface recognition.

Sumana Sharma1, S Josefin Bartholdson1, Amalie C M Couch1

  • 1Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.

Genome Research
|June 20, 2018
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This study introduces a genome-wide CRISPR screening method to identify cell surface receptor interactions. The approach successfully identified the insulin-like growth factor 2 receptor (IGF2R) as a direct binding partner for GABAB receptors.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Cell surface receptor interactions are crucial for cellular signaling but challenging to study biochemically due to transient nature and difficulty in solubilizing membrane proteins.
  • Existing biochemical assays often fail to capture the native conformation and transient interactions of membrane-embedded receptors.

Purpose of the Study:

  • To develop and validate a genome-wide, cell-based genetic screening approach using CRISPR gene knockout technology.
  • To overcome biochemical limitations in studying transient and membrane-embedded cell surface receptor interactions.
  • To identify genes and pathways involved in specific cell surface recognition events and functional receptor display.

Main Methods:

  • Genome-wide CRISPR gene knockout screening in a cell-based system.
  • Utilized high-affinity monoclonal antibodies and low-affinity ligands to identify interacting membrane-embedded receptors.
  • Optimized screening parameters, including glycocalyx contributions, for high statistical confidence in gene identification.

Main Results:

  • Successfully identified genes encoding directly interacting membrane-embedded receptors with high statistical confidence.
  • Discovered receptor-specific pathways essential for functional cell surface receptor display, including chaperones, post-translational modification enzymes, trafficking proteins, and transcription factors.
  • Identified insulin-like growth factor 2 receptor (IGF2R) as a direct binding partner for the R2 subunit of GABAB receptors, dependent on mannose-6-phosphate.

Conclusions:

  • The developed genome-wide screening approach effectively identifies molecular interactions and genetic pathways for cell surface receptor display.
  • This method reveals mechanisms for receptor internalization and signaling regulation, exemplified by the IGF2R-GABABR interaction.
  • The approach is versatile, applicable to receptors recognized by antibodies, secreted proteins, or membrane-bound ligands without prior biochemical assumptions.