Agonist regulation of the human platelet prostaglandin D2 receptor.

The effect of exposing platelets to prostaglandin D₂ (PGD₂) on hormone binding was studied. Incubation of platelets with PGD₂ for 2 hr resulted in a decrease in [³H]PGD₂ binding that was dose dependent. Inhibition of binding was 14% after incubation with 10^?8M PGD₂, 19% after incubation with 10^?7M PGD₂, and 40% after exposure to 10^?6M PGD₂. This decreased binding (desensitization) was specific for [³H]PGD₂ as binding to platelets by [³H]PGE₁ and the α-adrenergic antagonist [³H] dihydroergocryptine (DHEC) was comparable to control platelets. Saturation of [³]PGD₂ binding to desensitization platelets was at 27 fmole ligand/10⁸ platelets compared to 43 fmoles/10⁸ platelets in control platelets. Half-maximal saturation occured at 20 nM PGD₂ both for desensitized and control platelets, suggesting that decreased binding sites rather than altered affinity between ligand and receptor accounted for these results. These platelets had a partial increase in [³H]PGD₂ binding a few hours after plasma was washed free of PGD₂ with complete resensitization after 24 hr. Since prostaglandins such as PGI₂, PGD₂, and PGE₁ are potent inhibitors of platelet aggregation, decreased binding of platelets to these hormones after prostaglandin exposure may provide a mechanism for altered responsiveness of platelets to aggregating stimuli.     

Immunostimulating effect of D2 dopamine receptor agonist quinpirole

Immunostimulating activity of specific postsynaptic D2 receptor agonist quinpirole (Ly 171555) was studied in CBA mice. The drug was administered intraperitoneally in a dose of 1 mg/kg 30 min before the immunization with SRBC (5 x 10(8)). Considerable stimulation of rosette- and plaque-forming cell number was observed at the early stage (3-d day) and at more late stage of the immune response (5-th day). The increase of rosette- and plaque-forming cell number was provided by IgM-reaction enhancement.     

alpha 2A/alpha 2C-adrenergic receptor third loop chimera show that agonist interaction with receptor subtype backbone establishes G protein-coupled receptor kinase phosphorylation

The alpha(2A)-adrenergic receptor (AR) undergoes rapid agonist-promoted desensitization due to phosphorylation by G protein-coupled receptor kinases (GRKs) 2 and 3 at serines in the third intracellular loop of the receptor. In contrast, the alpha(2C)AR fails to display such desensitization or phosphorylation, which has been presumed to be due to this receptor lacking GRK phosphorylation sites. However, the alpha(2C)AR has multiple serines and threonines in putative favorable motifs within its third intracellular loop. We considered that the conformation of the third intracellular loop imposed by agonists binding to the transmembrane-spanning domains could be the basis of this subtype-specific property, rather than the presence or absence of phosphoacceptors per se. To address this, alpha(2A)/alpha(2C) third loop chimeric receptors were constructed. In whole cell phosphorylation studies, the alpha(2A) with the alpha(2C) third loop receptor underwent agonist-promoted phosphorylation while the alpha(2C) with the alpha(2A) third loop receptor did not, indicating that the agonist interaction with the parent receptor backbone establishes the phosphorylation phenotype. We postulated then that agonists with diverse structures that distinctly interact with alpha(2)AR should display different degrees of phosphorylation independent of receptor activation. Indeed, several full and partial agonists were identified, which evoked phosphorylation that was not related to intrinsic activity as established by [(35)S]guanosine 5'-3-O-(thio)triphosphate binding. Taken together, it appears that phosphorylation of the alpha(2)AR evoked by agonist is highly sensitive to the conformation of the third intracellular loop induced/stabilized by agonist to such an extent that these properties dictate the extent of phosphorylation of the loop when phosphoacceptors are present, and are the basis for subtype-specific phosphorylation.     

An antibody to dopamine D2 receptor inhibits dopamine antagonist and agonist binding to dopamine D2 receptor cDNA transfected mouse fibroblast cells.

A polyclonal antibody to dopamine D2 receptor (D2-receptor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [3H]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM09151-2 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 +/- 5.26 and 76.20 +/- 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltk- and Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB cells with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor.     

Decreased c-fos responses to dopamine D(1) receptor agonist stimulation in mice deficient for D(3) receptors.

The acute administration of dopamine D(1) receptor agonists induces the expression of the immediate early gene c-fos. In wild type mice, this induction is completely abolished by pretreatment with the D(1)-selective antagonist SCH23390, and pretreatment with the D(2)-like receptor antagonist eticlopride reduces the levels of c-fos expressed in response to D(1) receptor stimulation. Mice deficient for the dopamine D(3) receptor express levels of D(1) agonist-stimulated c-fos immunoreactivity that are lower than c-fos levels of their wild type littermates. Moreover, the acute blockade of D(2) receptors in D(3) mutant mice further reduces c-fos expression levels. These data indicate that the basal activity of both D(2) and D(3) receptors contributes to D(1) agonist-stimulated c-fos responses. The findings therefore indicate that not only D(2) but also D(3) receptors play a role in dopamine-regulated gene expression.     

Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins

The D(2) and D(3) receptors (D(2)R and D(3)R), which are potential targets for antipsychotic drugs, have a similar structural architecture and signaling pathway. Furthermore, in some brain regions they are expressed in the same cells, suggesting that differences between the two receptors might lie in other properties such as their regulation. In this study we investigated, using COS-7 and HEK-293 cells, the mechanism underlying the intracellular trafficking of the D(2)R and D(3)R. Activation of D(2)R caused G protein-coupled receptor kinase-dependent receptor phosphorylation, a robust translocation of beta-arrestin to the cell membrane, and profound receptor internalization. The internalization of the D(2)R was dynamin-dependent, suggesting that a clathrin-coated endocytic pathway is involved. In addition, the D(2)R, upon agonist-mediated internalization, localized to intracellular compartments distinct from those utilized by the beta(2)-adrenergic receptor. However, in the case of the D(3)R, only subtle agonist-mediated receptor phosphorylation, beta-arrestin translocation to the plasma membrane, and receptor internalization were observed. Interchange of the second and third intracellular loops of the D(2)R and D(3)R reversed their phenotypes, implicating these regions in the regulatory properties of the two receptors. Our studies thus indicate that functional distinctions between the D(2)R and D(3)R may be found in their desensitization and cellular trafficking properties. The differences in their regulatory properties suggest that they have distinct physiological roles in the brain.     

D5 dopamine receptor regulation of phospholipase D

D(1)-like receptors have been reported to decrease oxidative stress in vascular smooth muscle cells by decreasing phospholipase D (PLD) activity. However, the PLD isoform regulated by D(1)-like receptors (D(1) or D(5)) and whether abnormal regulation of PLD by D(1)-like receptors plays a role in the pathogenesis of hypertension are unknown. The hypothesis that the D(5) receptor is the D(1)-like receptor that inhibits PLD activity and serves to regulate blood pressure was tested using D(5) receptor mutant mice (D(5)(-/-)). We found that in the mouse kidney, PLD2, like the D(5) receptor, is mainly expressed in renal brush-border membranes, whereas PLD1 is mainly expressed in renal vessels with faint staining in brush-border membranes and collecting ducts. Total renal PLD activity is increased in D(5)(-/-) mice relative to congenic D(5) wild-type (D(5)(+/+)) mice. PLD2, but not PLD1, expression is greater in D(5)(-/-) than in D(5)(+/+) mice. The D(5) receptor agonist fenoldopam decreases PLD2, but not PLD1, expression and activity in human embryonic kidney-293 cells heterologously expressing the human D(5) receptor, effects that are blocked by the D(5) receptor antagonist SCH-23390. These studies show that the D(5) receptor regulates PLD2 activity and expression. The hypertension in the D(5)(-/-) mice is associated with increased PLD expression and activity. Impaired D(5) receptor regulation of PLD2 may play a role in the pathogenesis of hypertension.     

Impaired renal D(1)-like and D(2)-like dopamine receptor interaction in the spontaneously hypertensive rat

D(1)-like (D(1), D(5)) and D(2)-like (D(2), D(3), D(4)) dopamine receptors interact in the kidney to produce a natriuresis and a diuresis. Disruption of D(1) or D(3) receptors in mice results in hypertension that is caused, in part, by a decreased ability to excrete an acute saline load. We studied D(1)-like and D(2)-like receptor interaction in anesthetized spontaneously hypertensive rats (SHR) by the intrarenal infusion of Z-1046 (a novel dopamine receptor agonist with rank order potency of D(3)> or =D(4)>D(2)>D(5)>D(1)). Z-1046 increased glomerular filtration rate (GFR), urine flow, and sodium excretion in normotensive Wistar-Kyoto rats but not in SHRs. The lack of responsiveness to Z-1046 in SHRs was not an epiphenomenon, because intrarenal cholecystokinin infusion increased GFR, urine flow, and sodium excretion to a similar extent in the two rat strains. We conclude that renal D(1)-like and D(2)-like receptor interaction is impaired in SHRs. The impaired D(1)-like and D(2)-like receptor interaction in SHRs is not caused by alterations in the coding sequence of the D(3) receptor, the D(2)-like receptor expressed in rat renal tubules that has been shown to be involved in sodium transport. Because the diuretic and natriuretic effects of D(1)-like receptors are, in part, caused by an interaction with D(2)-like receptors, it is possible that the decreased Z-1046 action in SHRs is secondary to the renal D(1)-like receptor dysfunction in this rat strain.     

Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D(2)

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.     

Agonist-dependent interaction of the rat somatostatin receptor subtype 2 with cortactin-binding protein 1.

We report here an interaction between the C terminus of the rat somatostatin receptor subtype 2 (SSTR2) and a protein that has recently been identified as cortactin-binding protein 1 (CortBP1). Interaction is mediated by the PDZ (PSD-95/discs large/ZO-1) domain of CortBP1. As shown by in situ hybridization, SSTR2 and cortactin-binding protein are coexpressed in the rat brain. The association between SSTR2 and the PDZ-domain of CortBP1 was verified by overlay assays and by coprecipitation after transfection in human embryonic kidney (HEK) cells. Analysis by confocal microscopy indicates that CortBP1 is distributed diffusely throughout the cytosol in transfected cells and that it becomes concentrated at the plasma membrane when SSTR2 is present. This process is largely increased when the receptor is stimulated by somatostatin; as CortBP1 interacts with the C terminus of SSTR2, our data suggest that the binding of agonist to the receptor increase the accessibility of the receptor C terminus to the PDZ domain of CortBP1. Our data for the first time establish a link between a G-protein coupled receptor and constituents of the cytoskeleton.     

Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers

Hematopoietic cells uniquely express G(alpha16), a G protein alpha-subunit of the G(q)-type. G(alpha16) is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with G(alpha16). Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha16) and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.     

Evidence for alternative splicing of a dopamine D2 receptor in a teleost

Previous attempts at identifying an alternatively spliced dopamine (DA) D2 receptor in teleosts have proven unsuccessful. We provide evidence of a splicing event of a goldfish D2 (gfD2b1) receptor in the neuroendocrine brain of adult goldfish that produces a spliced short isoform (gfD2b1S). We also identify an additional novel D2b paralog (gfD2b2) that does not appear to be alternatively spliced in adult fish during the reproductive cycle. Relatively high mRNA levels of gfD2b1S were observed in the neuroendocrine brain and pituitary of sexually immature fish compared with sexually regressing fish. Real-time RT-PCR revealed that intraperitoneal injection of either SCH 23390 or sulpiride-D1- or D2-specific antagonists, respectively-decreased mRNA levels of gfD2b1S by 3.9-fold without affecting the unspliced isoforms. We suggest that the expression of the spliced D2 receptor modulates the inhibitory tone of DA throughout the reproductive cycle. The deduced amino acid sequence of gfD2b1S lacks 29 amino acids in the same region as the short isoform of mammalian D2. We propose that the gfD2b1S splice variant is the teleost ortholog of mammalian D2S. The hypothesis that D2 receptor splicing is a relatively recent innovation in higher tetrapods is not supported by our results.     

Evidence for a dopamine receptor subtype sensitive to combination of D1 with D2 antagonist in measurement of G-protein activity using rat striatal membranes

In rat striatal membranes, various kinds of dopamine receptor agonists stimulated low-Km GTPase activity in a concentration-dependent manner. This stimulation by bromocriptine, pergolide and apomorphine was partially inhibited by sulpiride (SUL), a D2-selective antagonist, markedly inhibited by combination of SUL with SCH 23390 (SCH), a D1-selective antagonist, and not modified by SCH alone. The stimulation by BAM-1110 was resistant to SUL or SCH alone but abolished by combination of SUL with SCH. These findings suggest the presence of another subtype of a dopamine receptor in a functional in vitro bioassay system in rat striata.     

D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.     

Evidence that calmodulin binding to the dopamine D2 receptor enhances receptor signaling

The Ca2+ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D2 receptor. We confirmed that the third intracellular loop of the D2 receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D2-like receptor agonist 7-OH-DPAT increased the colocalization of the D2 receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D2-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D2 receptor and CaM and also significantly decreased D2 receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D2 receptor enhances D2 receptor signaling.     

Dynamic interaction of human vasopressin/oxytocin receptor subtypes with G protein-coupled receptor kinases and protein kinase C after agonist stimulation

Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GRKs) and protein kinase C (PKC) are involved in their signal transduction. To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably expressed as green fluorescent protein fusion proteins and analyzed by fluorescence, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP tag did not interfere with ligand binding, internalization, and signal transduction. After agonist stimulation, PKC dissociated from the V(1)R, did not associate with the V(2)R, but associated with the V(3)R and the OTR. After AVP stimulation, only GRK5 briefly associated with AVPRs following a time course that varied with the receptor subtype. No GRK associated with the OTR. Exchanging the V(1)R and V(2)R C termini altered the time course of PKC and GRK5 association. Deletion of the V(1)R C terminus resulted in no PKC association and a ligand-independent sustained association of GRK5 with the receptor. Deletion of the GRK motif prevented association and reduced receptor phosphorylation. Thus, agonist stimulation of AVP/OT receptors leads to receptor subtype-specific interactions with GRK and PKC through specific motifs present in the C termini of the receptors.     

Purification of brain D2 dopamine receptor.

D2 dopamine receptors have been extracted from bovine brain using the detergent cholate and purified approximately 20,000-fold by affinity chromatography on haloperidol-sepharose and wheat germ agglutinin-agarose columns. The purified preparation contains D2 dopamine receptors as judged by the pharmacological specificity of [3H]spiperone binding to the purified material. The sp. act. of [3H]spiperone binding in the purified preparation is 2.5 nmol/mg protein. The purified preparation shows a major diffuse band at Mr 95,000 upon SDS-polyacrylamide gel electrophoresis and there is evidence for microheterogeneity either at the protein or glycosylation level. Photoaffinity labelling of D2 dopamine receptors also shows a species of Mr 95,000. The D2 dopamine receptor therefore is a glycoprotein of Mr 95,000.     

Synthesis and radioiodination of selective ligands for the dopamine D3 receptor subtype

Starting from FAUC 365, a series of iodine substituted heteroaryl carboxamides has been synthesized revealing high affinity and selectivity for the dopamine D3 receptor. Binding data showed a 15-560-fold selectivity for the dopamine D3 over D2. A 2,3-dichloro substitution pattern on the phenylpiperazine moiety led to the highest subtype selectivity, whereas the 2-methoxy substituted compounds showed superior D3 affinity. Suitable precursors were radioiodinated with high radiochemical yields (53-85%) leading to potential imaging agents for the D3 receptor by SPET.     

(G)n-Mononucleotide polymorphism in the human D4 dopamine receptor (DRD4) gene

Polymorphism in intron I of the human dopamine D4 receptor is described.