A Single Mutation at Lysine 241 Alters Expression and Trafficking of the D₂ Dopamine Receptor

Abstract

Ubiquitination of G protein-coupled receptors has been identified to regulate receptor signal transduction including agonist-induced internalization and sorting of internalized receptor for degradation or for recycling. Using co-immunoprecipitation and immunoblot analysis, I found that the membrane-associated D₂ dopamine receptor (DAR) is mono-ubiquitinated in the absence of an agonist following heterologous expression in human embryonic kidney cells (HEK293). By using site-directed mutagenesis, this report shows that the loss of lysine-241, K241A D₂ DAR reduced the amount of membrane-associated D₂ DAR. It is of interest that the K241A D₂ DAR also had a distinctly different ubiquitination pattern than the wild-type D₂ DAR. It is important to note that the ubiquitinated mutant D₂ DAR was degraded through ubiquitin-proteasome pathway. These data provide the factual evidence that a loss of lysine-241 of the D₂ DAR affects receptor ubiquitination and renders the protein susceptible to the proteasomal degradation.

Key words::

• D₂ dopamine receptor
• Ubiquitin
• Proteasomal degradation
• Mutation

ACKNOWLEDGMENTS

I thank Dr. Marie Wooten for Ub-HA construct and Dr. Rick Dobrowsky for helpful comments during preparation of this article. This research was supported by the University of Kansas General Research Fund allocation #2301813 to OJK.