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Role of c-Cbl carboxyl terminus in serotonin 5-HT2A receptor recycling and resensitization

Aleksander Baldys1, John R Raymond

  • 1Nephrology Division, Department of Medicine, Medical University of South Carolina, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425, USA.

The Journal of Biological Chemistry
|April 6, 2011
PubMed

Insights

The ubiquitin ligase c-Cbl is crucial for the recycling of the 5-hydroxytryptamine 2A receptor (5-HT(2A)R) to the cell surface. Its C-terminal region specifically regulates the timing and location of this essential receptor trafficking process.

Area of Science:

  • Cell Biology
  • Molecular Pharmacology
  • Biochemistry

Background:

  • The 5-hydroxytryptamine 2A receptor (5-HT(2A)R) is a G protein-coupled receptor known to internalize constitutively and upon agonist stimulation.
  • Understanding the precise mechanisms of G protein-coupled receptor endocytic sorting and recycling remains a significant challenge in cell biology.
  • The ubiquitin ligase c-Cbl has been previously identified as a key regulator of epidermal growth factor receptor vesicular sorting.

Purpose of the Study:

  • To investigate the potential role of the ubiquitin ligase c-Cbl in the recycling process of the 5-HT(2A)R.
  • To determine if c-Cbl influences the trafficking and resensitization of the 5-HT(2A)R after internalization.

Main Methods:

  • Demonstration of the physical association between 5-HT(2A)R and c-Cbl.
  • Down-regulation of c-Cbl expression using RNA interference to assess its impact on receptor recycling.
  • Immunofluorescence microscopy to visualize the localization of 5-HT(2A)R in cells expressing c-Cbl mutants.
  • Measurement of cytosolic calcium changes to evaluate receptor resensitization.

Main Results:

  • A direct association between 5-HT(2A)R and c-Cbl was confirmed.
  • Down-regulation of c-Cbl significantly impaired the efficient recycling of 5-HT(2A)R back to the plasma membrane.
  • Cells expressing c-Cbl mutants lacking carboxyl termini showed 5-HT(2A)R accumulation in sorting endosomes.
  • Mutant c-Cbl proteins exhibited reduced association with 5-HT(2A)R, correlating with delayed recycling and attenuated resensitization.

Conclusions:

  • The ubiquitin ligase c-Cbl plays a critical role in the recycling of the 5-HT(2A)R.
  • The C-terminal region of c-Cbl is essential for the temporal and spatial regulation of 5-HT(2A)R recycling.
  • These findings elucidate a novel mechanism governing G protein-coupled receptor trafficking and sorting.

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