Distinct Residues in the Carboxyl Tail Mediate Agonist-induced Desensitization and Internalization of the Human Dopamine D1 Receptor

Abstract

We have shown in a previous study that desensitization and internalization of the human dopamine D 1 receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D 1 receptor to study these processes. Mutant D 1 receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonistinduced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D 1 receptor. Dopamine D 1 receptors belong to the family of G proteincoupled receptors (GPCRs 1 ) and mediate their physiological response by coupling with stimulatory G proteins, which in turn activate adenylyl cyclase and increase levels of intracellular cAMP (1). Desensitization of GPCRs is defined as a loss of responsiveness to agonist treatment following prolonged agonist exposure (2, 3). In the dopamine D 1 receptor, we have shown that following short-term agonist exposure, desensitization and internalization were differentially regulated, both biochemically and temporally (4). Rapid desensitization results from receptor uncoupling from G protein following phosphorylation of intracellular domains of the receptor and subsequent internalization of receptor from the cell surface (5). The family of G protein-coupled receptor kinases (GRKs) and second messenger-dependent kinases, such as cAMP-dependent protein kinase (PKA), are serine/threonine kinases that function to uncouple the receptor from G protein (5-8). GRKs additionally promote the interaction of the receptor with arrestin proteins, which recruit the receptors to clathrin-coated pits for subsequent internalization (9).The ␤ 2 -adrenergic receptor is closely related structurally and functionally to the dopamine D 1 receptor, and the definition of the precise sites mediating desensitization occurred in a stepwise manner. Initial studies identified a region of the ␤ 2 -adrenergic receptor carboxyl tail that was involved in agonist-induced desensitization...

Regulation of Dopamine D1 Receptor Trafficking and Desensitization by Oligomerization with Glutamate N-Methyl-D-aspartate Receptors

“…The supernatant was centrifuged at 30,000 ϫ g for 20 min at 4°C. The resulting pellet was resuspended in 50 mM Tris-HCl containing 5 mM MgCl 2 , 1 mM EGTA, and the protease inhibitors (pH 7.8), layered on a 35% sucrose cushion, and centrifuged at 150,000 ϫ g for 90 min to separate the light vesicular and heavy membrane fractions as described by Lamey et al (26). The heavy fraction, at the bottom of the sucrose cushion, was resuspended in 50 mM Tris-HCl containing 5 mM EDTA, 1.5 mM CaCl 2 , 5 mM MgCl 2 , 5 mM KCl, and 120 mM NaCl (pH 7.4) and used for binding assay.…”

Dendritic distributions of dopamine D1 receptors in the rat nucleus accumbens are synergistically affected by startle-evoking auditory stimulation and apomorphine

“…Unlike D2Rs, D1Rs are predominantly located in distal dendrites and dendritic spines, which are major sites for synaptic plasticity (Delle Donne et al, 2004;Hara and Pickel, 2005). The surface expression of D1R is not stationary, but dynamically changed by synaptic activity and the availability of D1R agonists (Dumartin et al, 1998;Lamey et al, 2002). Auditory stimulation (AS), such as that used in acoustic startle testing, transiently decreases extracellular dopamine in the Acb (Humby et al, 1996).…”

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

“…However, the single substitution of T446 abolished agonist-induced receptor internalization without any significant effect on receptor desensitization (Lamey et al, 2002). These findings indicate that agonist-induced desensitization and internalization of D1R are regulated by separate and distinct residues within the carboxyl terminus of D1R.…”