Elimination of palmitoylation sites in the human dopamine D₁ receptor does not affect receptor-G protein interaction

Abstract

We have eliminated putative palmitoylation sites in the carboxyl tail of the human dopamine D₁ receptor by replacing the two cysteine residues with alanines either separately or together. The wild type and the three mutated dopamine D₁ receptors were stably expressed in baby hamster kidney cells and characterized to detect any resulting alterations in receptor-G protein interactions. The three mutant dopamine D₁ receptors retained the same proportion of high affinity state for agonists as wild type receptors and also no difference was observed in the stimulation of adenylyl cyclase activity. Our results are in contrast to those observed with the β₂-adrenoceptor and consistent with similar studies of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors, α₂-adrenoceptors, muscarinic M₂ receptors and thyrotropin releasing hormone (TRH) receptors. Thus, we suggest that palmitoylation appears to play a unique role in the β₂-adrenoceptors, and appears not to be essential in G protein coupling for the dopamine D₁ receptors. © 1997 Elsevier Science B.V. All rights reserved.

Introduction

Acylation by long-chain saturated fatty acids such as myristic and palmitic acids, is a common feature of membrane-associated proteins (Towler et al., 1988). Palmitoylation, a post-translational modification that initially takes place on nascent proteins in a post-endoplasmic reticulum compartment (Bonatti et al., 1989), is involved in important roles inside the cell, including anchorage of protein to the cell membrane, protein interaction with protein and determination of intracellular transportation/localization (Bizzozero et al., 1994). The alignment of the amino-acid sequences (Fig. 1) shows that the majority of members of the G-protein-coupled receptor superfamily have one or more cysteines located in the carboxyl tails near the seventh transmembrane domain. The receptors that have been shown to be palmitoylated via thioester bonds at these residues, are β₂-adrenoceptors (O'Dowd et al., 1989), rhodopsin (Ovchinnikov et al., 1988; Karnik et al., 1993), α_2A-adrenoceptors (Kennedy and Limbird, 1993), and the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors (Kawate and Menon, 1994).

Following our cloning of the human dopamine D₁ and the serotonin 5-HT_1Dβ receptors, we have demonstrated that both receptors and also the dopamine D_2L receptor are palmitoylated (Ng et al., 1993a, Ng et al., 1993b, Ng et al., 1994). Furthermore we demonstrated that dopamine D₁ receptors are palmitoylated in the absence of agonist and undergo an increase in palmitoylation that is agonist-induced (Ng et al., 1993a). It was demonstrated previously that the palmitoylation at Cys³⁴¹ in the carboxyl tail of β₂-adrenoceptors is important for receptor-G protein coupling, and that mutant β₂-adrenoceptors with Cys³⁴¹ replaced by glycine greatly reduced their ability to form the high affinity state for agonists and their ability to stimulate adenylyl cyclase activity (O'Dowd et al., 1989; Moffett et al., 1993). Subsequent to the initial experiments with β₂-adrenoceptors, substitutions of the equivalent cysteines on other receptors, including muscarinic M₂ (Van Koppen and Glaster, 1991), rhodopsin (Karnik et al., 1993), α_2A-adrenergic (Kennedy and Limbird, 1993), thyrotropin-releasing hormone (TRH) (Nussenzveig et al., 1993) and LH/hCG receptors (Kawate and Menon, 1994), showed that elimination of the palmitoylation site(s) did not perturb receptor-G protein coupling. However, palmitoylation has been implicated as being involved in sequestration and down-regulation of the α_2A-adrenergic (Eason et al., 1994), muscarinic M₂ (Van Koppen and Glaster, 1991), TRHR (Nussenzveig et al., 1993) and LH/hCG receptors (Kawate and Menon, 1994). These studies showed that prevention of palmitoylation had variable effects on G protein-coupled receptors.

The human dopamine D₁ receptor has an overall structure similar to the β₂-adrenoceptor and contains two putative palmitoylation sites (Cys³⁴⁷ and Cys³⁵¹) in the carboxyl tail (Fig. 2) (Sunahara et al., 1990). To clarify the role of palmitoylation in dopamine D₁ receptors, we eliminated these putative palmitoylation sites, both individually and together, using site-directed mutagenesis. We have found that substitution of either cysteine or both together did not affect ligand binding, receptor-G protein coupling, receptor desensitization, or agonist-induced stimulation of adenylyl cyclase activity.