Search

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.

Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway. We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP). Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC. Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation. While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes. This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.

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 wild-type 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.

Recently, we have shown that the dopamine D1B/D5 receptor displays binding and coupling properties that are reminiscent of those of the constitutively activated G protein-coupled receptors when compared with the related D1A/D1 receptor subtype (Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925-27931). The carboxyl-terminal region of the third cytoplasmic loop of several G protein-coupled receptors has been demonstrated to be important for the regulation of the equilibrium between inactive and active receptor conformations. In this cytoplasmic region, the primary structure of dopamine D1A and D1B receptors differs by only two residues: Phe264/Arg266 are present in D1A receptor compared with Ile288/Lys290 in the D1B receptor. To investigate whether these structural differences could account for the distinct binding and coupling properties of these dopamine receptor subtypes, we swapped the variant residues located in the carboxyl-terminal region by site-directed mutagenesis. The exchange of the D1A receptor residue Phe264 by the D1B receptor counterpart isoleucine led to a D1A receptor mutant exhibiting D1B-like constitutive properties. In contrast, substitution of D1B receptor Ile288 by the D1A receptor counterpart phenylalanine resulted in a loss of constitutive activation of the D1B receptor with binding and coupling properties similar to the D1A receptor. The Arg/Lys substitution had no effect on the function of either receptor. These results demonstrate that the carboxyl-terminal region, and in particular residue Ile288, is a major determinant of the constitutive activity of the dopamine D1B receptor. Moreover, these results establish that not only can agonist-independent activity of a receptor be induced, but when given the appropriate mutation, it can be reversed or silenced.

The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization.

Dopamine D1A and D1B receptor subtypes belong to the superfamily of G protein-coupled receptors. Both receptors are coupled to the activation of adenylyl cyclase and exhibit distinct brain distribution. To identify functional differences, binding and stimulation of adenylyl cyclase were assessed in 293 cells expressing transiently either dopamine D1A or D1B receptors. Membranes expressing D1B receptors displayed higher affinities for agonists than those expressing D1A receptors, whereas antagonist affinities were lower at the D1B than at the D1A receptor. Basal activity of adenylyl cyclase in whole 293 cells expressing various levels of D1B receptors was significantly higher than the basal activity measured in cells expressing D1A receptors. Maximal activation of adenylyl cyclase resulting from stimulation of the D1B receptor was less than that obtained following agonist activation of the D1A receptor. In cells expressing D1B receptors, agonists displayed an increased potency for stimulating adenylyl cyclase in comparison with the potencies determined for the D1A receptor. On the other hand, certain antagonists displayed "negative efficacy" at both receptor subtypes but had a more profound inhibition on the agonist-independent signaling activity of the D1B receptor. The properties described here are reminiscent of those of constitutively active G protein-coupled receptors obtained by site-directed mutations. Thus, the D1B receptor may represent a naturally occurring receptor subtype with properties akin to those of constitutively active G protein-coupled receptors. The different anatomical distribution and biochemical properties of these D1 receptors strengthen the functional distinctions between the two subtypes and could account for the basis of heterogeneity within a given class of neurotransmitter or hormone receptors. In addition, if these properties are recapitulated in cells expressing the D1B receptors, they may underlie important role in the regulation of physiological effects by dopamine. Finally, these results raise the interesting possibility that psychotropic antagonist drugs used in the management of certain brain disorders may have their beneficial actions as negative efficacy compounds.

An aromatic residue, tyrosine 326 in the prototypical human beta 2-adrenergic receptor, exists in a highly conserved sequence motif in virtually all members of the G protein-coupled receptor family. The potential role of this conserved aromatic amino acid residue in the cellular processes of sequestration (a rapid internalization of the surface receptor) and down-regulation (a slower loss of total cellular receptors) associated with agonist-mediated desensitization of the beta 2-adrenergic receptor was assessed by replacing tyrosine residue 326 with an alanine residue (beta 2AR-Y326A). This mutation completely abolishes agonist-mediated receptor sequestration without affecting the ability of the receptor to activate maximally adenylyl cyclase, to undergo rapid desensitization, and to down-regulate in response to agonist. The only other major change associated with the mutated receptor is a complete loss of the ability to resensitize following rapid desensitization. These results imply that this tyrosine residue, which is part of a highly conserved sequence motif in G protein-coupled receptors, may be responsible for their agonist-mediated sequestration and that sequestration and down-regulation of the receptor are dissociable phenomena. The lack of resensitization in the sequestration-defective beta 2-adrenergic receptor mutant strongly suggests that the sequestration pathway is an important mechanism by which cells re-establish the normal responsiveness of G protein-coupled receptors following the removal of agonist.