Abstract
Dopamine D2 and D3 receptors are similar subtypes with distinct interactions with arrestins; the D3 receptor mediates less agonist-induced translocation of arrestins than the D2 receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D2 and D3 receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin3 bound to glutathione transferase (GST) fusion proteins of the D2 receptor IC2 more avidly than to the D3 receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin3 to D2-IC2; arrestin binding to D2-IC2-K149C was greatly decreased compared with wild-type D2-IC2, whereas binding to the reciprocal mutant D3-IC2-C147K was enhanced compared with wild-type D3-IC2. Mutating this lysine in the full-length D2 receptor to cysteine decreased the ability of the D2 receptor to mediate agonist-induced arrestin3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D3 receptor increased receptor-mediated translocation of arrestin3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D2 receptor over the D3 receptor is due in part to Lys149, which could be exposed as a result of receptor activation.
Footnotes
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This work was supported by United States Public Health Service grants MH045372 (to K.A.N.), GM077561 (to V.V.G.), and EY011500 (to V.V.G.), and the VA Merit Review and Career Scientist programs (to K.A.N.).
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ABBREVIATIONS: IC2, second intracellular loop of a GPCR; IC3, third intracellular loop of a GPCR; GPCR, G protein-coupled receptor; GST, glutathione transferase; IC3, third intracellular loop of a GPCR; D2-IC2, second intracellular loop of the rat dopamine D2L receptor; D3-IC2, second intracellular loop of the rat dopamine D3 receptor; TBS, Tris-buffered saline; CMF-PBS, calcium- and magnesium-free phosphate-buffered saline.
- Received July 11, 2008.
- Accepted September 26, 2008.
- U.S. Government work not protected by U.S. copyright
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