The Role of Phosphorylation in D1 Dopamine Receptor Desensitization

Abstract

Homologous desensitization of D 1 dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D 1 receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.

Identification of a Novel Endocytic Recycling Signal in the D1 Dopamine Receptor

“…The specificity of this recycling defect is consistent with previous studies indicating that similar (or even more extensive) truncations of the D1 cytoplasmic tail do not disrupt ligand binding, G protein coupling, or rapid desensitization of receptors (10,25,26). Previous studies have identified a sequence in the proximal cytoplasmic tail of the D1 receptor that (by interacting with the cytoplasmic protein DRIP78) promotes export of newly synthesized receptors from the endoplasmic reticulum (32).…”

“…2A). Whereas previous work has shown that truncations near this region of the D1 receptor typically result in diminished amounts of surface receptor expression (25,26), cell clones used to compare trafficking of wild-type and mutant receptor constructs expressed the indicated receptors within a closely similar (ϳ3-fold) range. Furthermore, reduced recycling of the HA-D1-359T mutant receptor was observed in multiple independently isolated cell clones differing in mutant receptor expression by ϳ5-fold (data not shown).…”

Uncoupling and Endocytosis of 5-Hydroxytryptamine 4 Receptors

“…Thus, phosphorylation of the 5-HT 4 R C-terminal domain seems to be absolutely necessary for ␤-arrestin recruitment. This model contrasts with those of the dopamine D 1 receptor, in which phosphorylation of Ser/Thr permits access of arrestin to its receptor-binding domain rather than creating an arrestin-binding site per se (28). However, in our study, the ⌬346 mutant, whose i 3 loop is easily accessible, did not associate with ␤-arrestin, whereas the ⌬358 mutant, which possesses the Ser/Thr cluster, was always able to interact with ␤-arrestin.…”

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

“…Indeed, recent evidence has accumulated for a number of GPCRs suggesting that receptor phosphorylation is not always required for their desensitization or internalization (12)(13)(14)(15)(16)(17). This appears to be particularly true for G q/11 -linked GPCRs, which can be regulated by GRK2-mediated mechanisms that are independent of receptor phosphorylation.…”