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Related Experiment Videos

Type-specific sorting of G protein-coupled receptors after endocytosis.

P I Tsao1, M von Zastrow

  • 1Program in Cell Biology, Department of Biochemistry, University of California, San Francisco, California 94143-0984, USA.

The Journal of Biological Chemistry
|February 7, 2001
PubMed
Summary
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Role of endocytosis in signalling and regulation of G-protein-coupled receptors.

Biochemical Society transactions·2001

Distinct G protein-coupled receptors (GPCRs), like the beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR), show different trafficking after internalization. This differential sorting impacts receptor down-regulation, even with brief agonist exposure.

Area of Science:

  • Cell Biology
  • Molecular Pharmacology
  • Biochemistry

Background:

  • G protein-coupled receptors (GPCRs) mediate cellular responses to various stimuli.
  • Agonist binding triggers rapid internalization of receptors like beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) via clathrin-coated pits.
  • Receptor trafficking and down-regulation are critical for signal termination and cellular adaptation.

Purpose of the Study:

  • To investigate and compare the post-endocytic membrane trafficking of B2AR and DOR.
  • To determine the mechanisms underlying differential receptor down-regulation after agonist stimulation.
  • To elucidate the role of receptor sorting in agonist-induced desensitization.

Main Methods:

  • Stable transfection of HEK293 cells with B2AR and DOR.

Related Experiment Videos

  • Radioligand binding assays and immunoblotting to quantify functional and total receptor levels.
  • Flow cytometry and surface biotinylation assays to track receptor localization.
  • Treatment with lysosomal proteolysis inhibitors.

    • Main Results:

    • B2AR exhibited minimal (<10%) down-regulation, while DOR showed substantial (>=50%) down-regulation after 3 hours of continuous agonist incubation.
    • Differential sorting of B2AR (recycling) and DOR (non-recycling) was detected within 10 minutes post-endocytosis, preceding receptor degradation.
    • DOR degradation was sensitive to lysosomal inhibitors, and later degradation steps did not require continuous agonist presence.

    Conclusions:

    • Distinct GPCRs, despite sharing internalization mechanisms, exhibit significantly different endocytic trafficking pathways.
    • Early sorting events dictate the subsequent fate of internalized receptors, influencing down-regulation.
    • Brief agonist application can lead to substantial receptor down-regulation due to differential trafficking and sorting.