N-linked Glycosylation on the N-terminus of the dopamine D2 and D3 receptors determines receptor association with specific microdomains in the plasma membrane

• Cell surface expression of D2 and D3 receptor is commonly regulated by glycosylation.
• Desensitization of D3 but not D2 receptor is regulated by glycosylation.
• Glycosylation of D2 and D3 receptor exerts opposite effects on receptor endocytosis.
• Glycosylation of D3 receptor on the N-terminus and extracellular loops has different roles.
• Glycosylation on the N-terminus directs selection of endocytic pathways.

Numerous G protein-coupled receptors (GPCRs) are glycosylated at extracellular regions. The regulatory roles of glycosylation on receptor function vary across receptor types. In this study, we used the dopamine D2 and D3 receptors as an experimental model to understand the underlying principles governing the functional roles of glycosylation. We used the pharmacological inhibitor, tunicamycin, to inhibit glycosylation, generated chimeric D2 and D3 receptors by swapping their respective N-termini, and produced the glycosylation site mutant D2 and D3 receptors to study the roles of glycosylation on receptor functions, including cell surface expression, signaling, and internalization through specific microdomains. Our results demonstrate that glycosylation on the N-terminus of the D3 receptor is involved in the development of desensitization and proper cell surface expression. In addition, glycosylation on the N-terminus mediates the internalization of D2 and D3 receptors within the caveolae and clathrin-coated pit microdomains of the plasma membrane, respectively, by regulating receptor interactions with caveolin-1 and clathrin. In conclusion, this study shows for the first time that glycosylation on the N-terminus of GPCRs is involved in endocytic pathway selection through specific microdomains. These data suggest that changes in the cellular environment that influence posttranslational modification could be an important determinant of intracellular GPCR trafficking.

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