Delineation of the Structural Basis for the Activation Properties of the Dopamine D1 Receptor Subtypes

Abstract

To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wildtype D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.The classical paradigm for G protein-coupled receptor (GPCR) 1 activation is described by the binding of an agonist to an inactive receptor state (R). This process leads to the formation of an active receptor state (R*) which interacts with heterotrimeric GTP-binding proteins (G proteins) to initiate a variety of intracellular signaling events. In recent years, however, mutagenesis studies have led to a notion asserting that GPCRs exist in an equilibrium between two interchangeable conformational states, R and R* (1-3). In the absence of ligand (agonist), GPCRs are predominantly maintained in an inactive R state by intramolecular constraints that prohibit the interaction with G proteins. These intramolecular constraints are released upon agonist binding or by mutations. Indeed, mutations in the carboxyl end of the third cytoplasmic loop of GPCRs can result in mutant receptors displaying high ...

Insight into the Mechanism of Dopamine D1-like Receptor Activation

“…To narrow down the structural determinants within TRL involved in the subtype-specific ligand binding and G protein activation properties of the D1-like receptors, additional mutant receptors were constructed in which either the EL3 (10) or CT sequences (11) were swapped between the D1A and D1B receptors. Chimeric receptors harboring EL3 of their respective wild-type counterparts exhibited a complete reversal of agonist-mediated maximal activation of AC, whereas DA affinity and constitutive activity of the chimeras were only partially modulated by the exchange (10). In contrast, chimeric receptors harboring the CT of their respective wild-type counterparts displayed a full switch in DA affinity and constitutive activity, whereas DA potency decreased and agonist-mediated maximal activation of AC increased for both chimeras (11).…”

“…Previously, we have shown that a structural domain (referred to as the terminal receptor locus (TRL)) encompassing the sixth and seventh transmembrane regions (TM6 and TM7), third extracellular loop (EL3) and cytoplasmic tail (CT) plays an important role in the phenotypic expression of ligand binding and G protein coupling properties of D1-like receptors (10). To narrow down the structural determinants within TRL involved in the subtype-specific ligand binding and G protein activation properties of the D1-like receptors, additional mutant receptors were constructed in which either the EL3 (10) or CT sequences (11) were swapped between the D1A and D1B receptors.…”

“…Whole Cell cAMP Assay-Regulation of AC activity by wild-type and chimeric receptors was assessed using a whole cell cAMP assay as described previously (10,11). Following overnight incubation with the DNA-calcium phosphate precipitate, HEK 293 cells were reseeded in 6-or 12-well dishes.…”

“…Construction of Chimeric Human D1A and D1B Receptors-To construct chimeric receptors harboring only the EL3 and CT regions of their wild-type counterparts, we took advantage of existing chimeras in which the EL3 region was swapped between the D1A and D1B subtypes (10). We have utilized these chimeras (D1A-EL3B and D1B-EL3A) and wild-type receptors as templates to engineer two additional chimeric receptors (D1A-EL3CTB and D1B-EL3CTA) in which the CT regions were exchanged by gene splicing using a PCR-based overlap extension approach.…”

神经细胞中多巴胺d1和d5受体不同亚细胞分布和胞内转运的特性

“…In a systematic binding study of the C-termini of all dopamine receptor subtypes, D5R but not D1R C-terminus selectively and strongly bound to a membrane-associated protein sorting nexin 1 (SNX1) [26] . In addition, the C-termini of D1-like receptors also governed their distinct binding affinity to agonists [27,28] . Swapping the C-termini of D1R and D5R resulted in the full switch of the affinities.…”