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Related Experiment Videos

Internalization of type-A endothelin receptor.

J Wang1, W J Chiou, G D Gagne

  • 1Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois, USA.

Journal of Cardiovascular Pharmacology
|November 15, 2000
PubMed
Summary

Endothelin-1 (ET-1) binding triggers the internalization of endothelin ET(A) receptors in CHO cells. Internalized receptors traffic to perinuclear structures and may recycle to the cell surface.

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Area of Science:

  • Molecular pharmacology
  • Cell biology
  • Receptor trafficking

Background:

  • Endothelins (ETs) are peptides that bind to ET(A) and ET(B) receptors, mediating various physiological responses.
  • Understanding receptor internalization is crucial for comprehending cellular signaling pathways.

Purpose of the Study:

  • To investigate the internalization process of the ET(A) receptor in Chinese hamster ovary (CHO) cells.
  • To determine the localization and fate of internalized ET(A) receptors and ET-1.

Main Methods:

  • Stable transfection of CHO cells with ET(A) receptor cDNA.
  • Immunostaining using polyclonal antibodies to localize receptors.
  • Treatment with biotinylated ET-1 and Texas Red-labeled streptavidin to track ligand-receptor complexes.

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  • Use of protein kinase inhibitors (KT5926, staurosporine) to assess pathway involvement.

    • Main Results:

    • ET-1 binding induced ET(A) receptor internalization in a time-dependent manner (40% internalized after 30 min at 37°C).
    • Internalized receptors localized to perinuclear structures after 60 min of ET-1 treatment.
    • ET-1 remained bound to the receptor for up to 60 min before dissociation, suggesting potential receptor recycling and ligand degradation.
    • Protein kinase inhibitors partially inhibited ET(A) receptor internalization.

    Conclusions:

    • ET-1 binding initiates a time-dependent internalization of ET(A) receptors in CHO cells.
    • Internalized ET(A) receptors accumulate in perinuclear regions and may subsequently recycle.
    • The internalization process is partially regulated by protein kinase pathways.