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

Abstract

Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D(2) and D(3) 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. The N-terminal region of the D(2) receptor was gradually shortened or switched with that of the D(3) 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. Shortening the N-terminal region of the D(2) 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 D(2) and D(3) receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D(2) receptors. Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D(2) receptors. The N-terminal region of the D(2) 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.