The N‐terminal region of the dopamine D₂ receptor, a rhodopsin‐like GPCR, regulates correct integration into the plasma membrane and endocytic routes

Abstract

BACKGROUND AND PURPOSEFunctional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACHThe N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTSShortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONSShortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties.

The N-terminus of the yeast G protein-coupled receptor Ste2p plays critical roles in surface expression, signaling, and negative regulation

“…Similar results were observed with the N-terminal region of the dopamine D2 receptor and the neuropeptide Y subfamily receptors whereby truncation decreased cell surface expression of these receptors [53, 54]. However, N-terminal truncations do not always result in poor plasma membrane localization.…”

Dopamine-2 receptor extracellular N-terminus regulates receptor surface availability and is the target of human pathogenic antibodies from children with movement and psychiatric disorders

“…To further refine the specific antigenic region within residues 23-37 of D2R, recognised by two-thirds of our MPD cohort, we next examined the effect of N-glycosylation as it has been reported to influence D2R trafficking to the cell membrane [11]. Indeed, targeted mutagenesis of all three putative N-glycosylation sites (N5Q/N17Q/N23Q, Additional file 1: Figure S4a) led to low cell surface expression and intracellular sequestration of the mutant (Additional file 1: Figure S4).…”

Serum IgE clearance is facilitated by human FcεRI internalization

“…One of these may laterally interact with FcεRI through carbohydrate moieties attached to the FcεRIα extracellular domain, thus driving FcεRI to endocytic pathways. Glycosylation of G protein-coupled receptors has been shown to influence the kinetics or routing of endocytosis (43,44). Notably, FcεRI in BDCA1 + DCs appears to be glycosylated distinctly from FcεRI in basophils, as the hFcεRI immunoprecipitated from these DC lysates mobilized, according to SDS-PAGE, at a slightly faster rate.…”