Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

Abstract

The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D 1 receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D 1 receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D 1 -EGFP). D 1 -EGFP was similar to the untagged D 1 receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D 1 -EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D 1 receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D 1 receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.

Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons

“…These results are consistent with previous work demonstrating agonist-induced internalization of the D1 receptor in heterologous expression systems (Oakley et al 2000;Gardner et al 2001;Mason et al 2002).…”

“…Class B receptors remain associated with arrestin during internalization, which makes the recycling and dephosphorylation of the receptor occur more slowly, and have a similar affinity for arrestin2 and 3 (Shenoy and Lefkowitz 2003). The dopamine D1 receptor has been designated a class A GPCR, along with the b 2 -adrenoceptor, whereas the neurokinin NK-1 and vasopression V2 receptors are examples of class B GPCRs (Oakley et al 2000).Desensitization and internalization of the dopamine D1 receptor have been described in a variety of cell lines and tissue preparations (Ng et al 1994;Tiberi et al 1996;Gardner et al 2001;Lamey et al 2002;Mason et al 2002;Kim et al 2004). Desensitization of the D1 receptor may depend on phosphorylation of the receptor by both GPCR kinase-and second messenger-dependent protein kinases such as protein kinase Abbreviations used: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; D1-CT, C-terminus of the dopamine D1 receptor; D1-IC3, third cytoplasmic loop of the dopamine D1 receptor; GPCR, G protein-coupled receptor; GST, glutathione-S-transferase; PBS, phosphate-buffered saline.…”

“…Desensitization and internalization of the dopamine D1 receptor have been described in a variety of cell lines and tissue preparations (Ng et al 1994;Tiberi et al 1996;Gardner et al 2001;Lamey et al 2002;Mason et al 2002;Kim et al 2004). Desensitization of the D1 receptor may depend on phosphorylation of the receptor by both GPCR kinase-and second messenger-dependent protein kinases such as protein kinase Abbreviations used: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; D1-CT, C-terminus of the dopamine D1 receptor; D1-IC3, third cytoplasmic loop of the dopamine D1 receptor; GPCR, G protein-coupled receptor; GST, glutathione-S-transferase; PBS, phosphate-buffered saline.…”

Protein Kinase D1-Dependent Phosphorylation of Dopamine D1 Receptor Regulates Cocaine-Induced Behavioral Responses

“…In general, GRKs mediate agonist-induced phosphorylation of D1R, whereas PKA and PKC contribute to heterologous receptor phosphorylation (Gainetdinov et al, 2004). PKA can phosphorylate T268 in the third intracellular loop and T380 in the carboxyl terminus of D1R, which regulates either the rate of desensitization or intracellular trafficking of the receptor once internalized (Jiang and Sibley, 1999;Mason et al, 2002). Constitutive or heterologous PKC (including PKCa, bI, g, d, and e) phosphorylates D1R and dampens DA activation of the receptor, thus attenuating D1R-mediated signaling (Gardner et al, 2001;Rankin and Sibley, 2010;Rex et al, 2008;Rex et al, 2010).…”

Disruption of dopamine D1 receptor phosphorylation at serine 421 attenuates cocaine-induced behaviors in mice

“…In addition to GRKs, the second-messenger activated protein kinases, PKA and PKC, can also phosphorylate the D1R. For example, PKA phosphorylates T268 in the third cytoplasmic loop and T380 in the carboxylterminus of the D1R, which regulates either the rate of desensitization or the intracellular traffi cking of the receptor once internalized [23,24] . D1R can also be phosphorylated either constitutively or heterologously by PKC (including PKC α, βI, γ, δ, and ε) at S259 in the 3 rd intracellular loop and S397, S398, S417, and S421 in the carboxyl terminus, which results in the inhibition of receptor-G-protein coupling and downstream signaling [25][26][27] .…”