The role of phosphorylation in D-1 dopamine receptor desensitization - Evidence for a novel mechanism of arrestin association

Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1406, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2004; 279(9):7999-8010. DOI: 10.1074/jbc.M308281200
Source: PubMed


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.

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Available from: Daniel B Williams, Jan 23, 2016
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    • "This data suggested that this C-terminal di-L motif is a plasma membrane targeting signal. Kim et al. [25] reported that the cell surface expression of a deletion mutant D1R (truncated at position 347) was diminished relative to the wild-type. In their mutant, di-L motif was intact, but the mutant could not get to the cell surface. "
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    • "These and similar data obtained with a truncated opioid receptor [53] or with the leukotriene B4 receptor 1 (BLT1) [54] do not support a general role for receptor phosphorylation in the control of GPCRs internalization. Overall, these findings led Kim et al. to suggest that the role of receptor phosphorylation allow the access of arrestin to its receptor binding domain than to create an arrestin-binding site [55]. "
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    • "The binding of arrestin to GPCRs involves both phosphorylation-dependent and independent mechanisms. Phosphorylation of receptor intracellular domains by GPCR kinases may create a binding site for positively charged arrestin residues (Gurevich and Gurevich, 2006) and may also cause the intracellular domains to undergo conformational changes exposing nonphosphorylated residues that participate in forming the arrestin binding site (Kim et al., 2004). Receptor activation This work was supported by United States Public Health Service grants MH045372 (to K "
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