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Control of CCR5 Cell-Surface Targeting by the PRAF2 Gatekeeper.

Elisa Da Silva1, Mark G H Scott1, Hervé Enslen1

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PRAF2 acts as a gatekeeper, retaining chemokine receptor CCR5 in the ER. This protein- G protein-coupled receptor interaction prevents CCR5 export to the cell surface, opposing the role of escort protein CD4.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cell-surface protein transport relies on ER export, regulated by escort and gatekeeper proteins.
  • G protein-coupled receptors (GPCRs) are crucial for cellular signaling and drug targeting.
  • PRAF2 is known to retain GABAB receptor subunits in the ER.

Purpose of the Study:

  • To investigate the role of PRAF2 in the ER export of the chemokine receptor CCR5.
  • To elucidate the interaction mechanism between PRAF2 and CCR5.
  • To compare the functions of PRAF2 and CD4 in CCR5 trafficking.

Main Methods:

  • Bioluminescence Resonance Energy Transfer (BRET) for subcellular localization and proximity assays.
  • Co-immunoprecipitation (Co-IP) to confirm protein interactions.
  • Site-directed mutagenesis to study specific motifs within CCR5.

Main Results:

  • PRAF2 inhibits CCR5 export to the plasma membrane in a concentration-dependent manner.
  • PRAF2 interacts with CCR5's transmembrane domains, not its C-terminal tail.
  • CCR5's potential retention motif mutation impairs CD4 interaction but not PRAF2 binding.
  • CD4 promotes CCR5 export, acting antagonistically to PRAF2.

Conclusions:

  • PRAF2 functions as a gatekeeper for CCR5, preventing its ER exit.
  • The interaction between PRAF2 and CCR5 involves transmembrane domains.
  • PRAF2 and CD4 have opposing roles in regulating CCR5 trafficking to the cell surface, likely at the COPII vesicle recruitment stage.