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G protein independent phosphorylation and internalization of the delta-opioid receptor

Faye A Bradbury1, Jennifer C Zelnik, John R Traynor

  • 1Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-5632, USA.

Journal of Neurochemistry
|April 7, 2009
PubMed

Insights

Pertussis toxin (PTX) treatment did not fully block delta-opioid receptor internalization, suggesting G protein-independent pathways contribute to this process. Agonist-induced phosphorylation at Ser363 predicted receptor internalization.

Area of Science:

  • Pharmacology
  • Cell Biology
  • Molecular Biology

Background:

  • Delta-opioid receptor (DOR) activation triggers internalization.
  • This process involves G protein-coupled receptor kinase-2 (GRK2), beta-arrestin, and clathrin-coated pits.
  • Pertussis toxin (PTX) inhibits G protein signaling.

Purpose of the Study:

  • To investigate the role of G protein signaling in DOR internalization.
  • To determine if PTX treatment blocks DOR phosphorylation and beta-arrestin recruitment.
  • To assess the impact of PTX on agonist-induced DOR internalization.

Main Methods:

  • Human embryonic kidney cells expressing DOR were treated with PTX.
  • Cells were stimulated with various DOR agonists.
  • Receptor phosphorylation at Ser363, beta-arrestin 2-green fluorescent protein recruitment, and receptor internalization were measured.
  • G protein activation was assessed via guanosine-5'-O-(3-35S-thio)triphosphate binding.

Main Results:

  • PTX treatment did not prevent DOR phosphorylation at Ser363 or beta-arrestin 2 recruitment.
  • PTX only partially inhibited internalization induced by [D-Pen2, D-Pen5]enkephalin, deltorphin II, [Met5]enkephalin, and BW373U86.
  • Morphine, oxymorphindole, and TAN-67 did not induce internalization, despite TAN-67 activating G protein.
  • Agonist-induced Ser363 phosphorylation correlated with internalization capacity.

Conclusions:

  • G protein signaling plays a role in DOR internalization, but is not essential.
  • Alternative, G protein-independent pathways mediate DOR internalization.
  • Receptor phosphorylation at Ser363 is a key event predicting internalization.

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