Insight into the Mechanism of Dopamine D1-like Receptor Activation

Abstract

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors. However, the D1A-CTB chimera, unlike the D1B subtype, exhibits a significantly lower DA potency for stimulating adenylyl cyclase and a drastically lower maximal binding capacity (Bmax). Here, using a functional complementation of chimeric D1-like receptors, we have identified the human D1B receptor regions regulating the intramolecular relationships that lead to an increased DA potency and contribute to Bmax. We demonstrate that the addition of variant residues of the third extracellular loop (EL3) of the human D1B receptor into D1A-CTB chimera leads to a constitutively active mutant receptor displaying an increased DA affinity, potency, and Bmax. These results strongly suggest that constitutively active D1-like receptors can adopt multiple active conformations, notably one that confers increased DA affinity with decreased DA potency and Bmax and another that imparts increased DA affinity with a strikingly increased DA potency and Bmax. Overall, we show that a novel molecular interplay between EL3 and CT regulates multiple active conformations of D1-like receptors and may have potential implications for other G protein-coupled receptor classes. Dopamine (DA)1 elicits its physiological and endocrine effects through the interaction with D1-like and D2-like G protein-coupled receptor (GPCR) subfamilies (1). D1-like receptors couple to the activation of adenylyl cyclase (AC) and in mammals are further divided into D1A (or D1) and D1B (or D5) subtypes. The D1B receptor distinguishes itself from the D1A subtype by a higher constitutive activity, increased affinity and potency for agonists, decreased affinity for antagonists, and a lower agonist-mediated maximal activation (2). Recently, a study highlighted the potential physiological relevance of the D1B receptor constitutive activity (3). Indeed, the extent of the D1B receptor constitutive activity controls the estrogen-induced mRNA expression of atrial natriuretic factor in primary hypothalamic neurons, a neuroendocrine process that potentially plays a role in D1B receptor-mediated facilitation of female sexual behavior (3). Moreover, the detection of D1-like subtype mRNA expression and/or activity in human breast and neuroendocrine gastrointestinal tumor cells (4, 5) underscores the importance of these receptors as well as, potentially, their levels of constitutive activation in regulating DA function in pathophysiological conditions, as shown previously with other constitutively active mutant GPCRs (6). Thus, D1A and/or D1B subtype-specific ligands may help in the treatment of pathologies for which compromised D1-like receptor responsiveness is purported (7-9). However, the structure-function relationships shaping the functional properties of the D1-like receptors remain unclear.Previously, we have shown that a structural domain (referred to...

HCO3−/Cl− Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

“…A full-length version of the human dopamine D1 receptor (hD1) with the cytoplasmic tail fused to GFP (hD1-GFP) was subcloned into cDNA3.1 (Invitrogen) and the sequence confirmed by direct sequencing. Plasma membrane localization and functionality of hD1-GFP was validated in transfected human embryonic kidney 293 (HEK293) cells using radioligand binding, whole cell cAMP assays and indirect immunofluorescence microscopy (data not shown) as previously described [42], [43]. In all cases, cRNA was prepared from linearized cDNA templates (mMESSAGE mMACHINE; Ambion, Austin, TX) resuspended in RNAse-free water and stored at −80°C until microinjected.…”

The Role of Phosphorylation in D1 Dopamine Receptor Desensitization

“…Although there are currently no physicochemical data demonstrating this, Tiberi and co-workers (15,(23)(24)(25) have provided evidence consistent with the association of these two receptor domains. By using receptor chimeras and truncation mutants, these investigators have shown that the carboxyl termini of the D 1 -like receptors (D 1 and D 5 ) play critical roles in determining agonist affinity, G protein coupling, and constitutive activity of the receptors (15,(23)(24)(25). Because it is unlikely that these functional effects are directly controlled by the carboxyl termini and it is known that the 3rd cytoplasmic loop is critical for D 1 receptor coupling to G s (26) and also that the 3rd loop of the related D 3 receptor controls receptor affinity for agonists (27), the simplest explanation is the carboxyl terminus influences 3rd loop functions through direct domain-domain interactions.…”

Role of the fourth intracellular loop of D1‐like dopaminergic receptors in conferring subtype‐specific signaling properties

“…Transfected HEK293 cells were washed with phosphate‐buffered saline (PBS), trypsinized, reseeded in 150‐mm dishes and grown for an additional 48 h. Crude membrane preparations expressing wild‐type or chimeric receptors were prepared as previously described [18]. Membrane preparations were either used immediately (saturation studies) or frozen in liquid nitrogen and stored at −80 °C until needed (competition studies).…”

“…Competition studies using DA were done in the presence of 0.1 mM ascorbic acid (dissolved in milli‐Q‐water). Membranes were incubated for 90 min at room temperature and binding assays stopped using rapid filtration through glass fiber filters (GF/C, Whatman) as described before [18]. Radioactivity bound to filters was measured using liquid scintillation counting (Beckman Counter, LS 6500).…”