Absence of Cytosolic 2-Cys Prx Subtypes I and II Exacerbates TNF-α-Induced Apoptosis via Different Routes.

There are abundant peroxiredoxin (Prx) enzymes, but an increase of cellular H2O2 level always happens in apoptotic cells. Here, we show that cellular H2O2 switches different apoptosis pathways depending on which type of Prx enzyme is absent. TNF-α-induced H2O2 burst preferentially activates the DNA damage-dependent apoptosis pathway in the absence of PrxI. By contrast, the same H2O2 burst stimulates the RIPK1-dependent apoptosis pathway in the absence of PrxII by inducing the destruction of cIAP1 in caveolar membrane. Specifically, H2O2 induces the oxidation of Cys308 residue in the cIAP1-BIR3 domain, which induces the dimerization-dependent E3 ligase activation. Thus, the reduction in cIAP level by the absence of PrxII triggers cell-autonomous apoptosis in cancer cells and tumors. Such differential functions of PrxI and PrxII are mediated by interaction with H2AX and cIAP1, respectively. Collectively, this study reveals the distinct switch roles of 2-Cys Prx isoforms in apoptosis signaling.

Silencing of peroxiredoxin II by promoter methylation is necessary for the survival and migration of gastric cancer cells.

Peroxiredoxin (Prx), a family of ubiquitous thiol peroxidases, functions as a redox signaling regulator that controls cellular H2O2 in mammalian cells and has recently received attention for being overexpressed in various cancer types. In this study, we show that Prx type II (PrxII) is rather silenced in gastric cancer cells. PrxII expression is severely downregulated in 9 out of the 28 gastric cancer cell lines. Strikingly, PrxII expression is completely lost in three cell lines, MKN28, MKN74 and SNU484. Loss of PrxII expression is due to DNA methyltransferase 1-dependent methylation at the promoter region of the PrxII gene. Restoration of PrxII expression using a retroviral system markedly reduces the colony-forming ability and migratory activity of both MKN28 and SNU484 cells by inhibiting Src kinase. Mechanistically, PrxII peroxidase activity is essential for regulating gastric cancer cell migration. Bioinformatics analysis from The Cancer Genome Atlas stomach cancer data (STAD) revealed significantly low PrxII expression in gastric cancer patients and a negative correlation between PrxII expression and methylation levels. More importantly, low PrxII expression also strongly correlates with poor survival in cancer patients. Thus our study suggests that PrxII may be the first thiol peroxidase that simultaneously regulates both survival and metastasis in gastric cancer cells with high clinical relevance.

Molecular Signature That Determines the Acute Tolerance of G Protein-Coupled Receptors.

Desensitization and acute tolerance are terms used to describe the attenuation of receptor responsiveness by prolonged or intermittent exposure to an agonist. Unlike desensitization of G protein-coupled receptors (GPCRs), which is commonly explained by steric hindrance caused by the ß-arrestins that are translocated to the activated receptors, molecular mechanisms involved in the acute tolerance of GPCRs remain unclear. Our studies with several GPCRs and related mutants showed that the acute tolerance of GPCRs could occur independently of agonist-induced ß-arrestin translocation. A series of co-immunoprecipitation experiments revealed a correlation between receptor tolerance and interactions among receptors, ß-arrestin2, and Gßγ. Gßγ displayed a stable interaction with receptors and ß-arrestin2 in cells expressing GPCRs that were prone to undergo tolerance compared to the GPCRs that were resistant to acute tolerance. Strengthening the interaction between Gßγ and ß-arrestin rendered the GPCRs to acquire the tendency of acute tolerance. Overall, stable interaction between the receptor and Gßγ complex is required for the formation of a complex with ß-arrestin, and determines the potential of a particular GPCR to undergo acute tolerance. Rather than turning off the signal, ß-arrestins seem to contribute on continuous signaling when they are in the context of complex with receptor and Gßγ.

RalA employs GRK2 and ß-arrestins for the filamin A-mediated regulation of trafficking and signaling of dopamine D2 and D3 receptor.

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D2 and D3 receptors (D2R, D3R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D2R and D3R, comparative studies were conducted on the signaling and intracellular trafficking of the D2R and D3R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D2R and D3R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D2R and D3R, through GRK2 and ß-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D2R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D2R. Knockdown of FLNA or coexpression of active RalA prevented D3R from coupling with G protein. The selective involvement of GRK2- and ß-arrestins in the RalA-mediated cellular processes of the D2R and D3R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D2R and D3R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and ß-arrestins.

Functional Regulation of Dopamine D3 Receptor through Interaction with PICK1.

PICK1, a PDZ domain-containing protein, is known to increase the reuptake activities of dopamine transporters by increasing their expressions on the cell surface. Here, we report a direct and functional interaction between PICK1 and dopamine D3 receptors (D3R), which act as autoreceptors to negatively regulate dopaminergic neurons. PICK1 colocalized with both dopamine D2 receptor (D2R) and D3R in clusters but exerted different functional influences on them. The cell surface expression, agonist affinity, endocytosis, and signaling of D2R were unaffected by the coexpression of PICK1. On the other hand, the surface expression and tolerance of D3R were inhibited by the coexpression of PICK1. These findings show that PICK1 exerts multiple effects on D3R functions.

Agonist-induced changes in RalA activities allows the prediction of the endocytosis of G protein-coupled receptors.

GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gßγ. These results suggest that agonist-induced Gßγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs.

Design, synthesis and evaluation of bitopic arylpiperazinephenyl-1,2,4-oxadiazoles as preferential dopamine D3 receptor ligands.

The dopamine D3 receptor (D3R) was proposed as a therapeutic target for drug development to treat drug abuse and addiction and neuropsychiatric disorders. Several D3R-selective modulators over the dopamine D2 receptor (D2R) can avoid extrapyramidal symptoms (EPS) and hyperprolactinemia. However, few biased D3R ligands were identified or showed a narrow range of selectivity at the D3R over D2R because of their high sequence homology. Herein, we designed, synthesized and evaluated the binding affinity of a series of bitopic ligands: arypiperazine-phenyl-1,2,4-oxadiazoles. Compound 9e·HCl was the most potent and selective D3R modulator among these bitopic ligands. Molecular modeling revealed that D3R selectivity depends on the divergence of secondary binding pocket (SBP) in D3R and D2R. Specifically, non-conserved Tyr36, EL1 especially non-conserved Thr92 and Gly94, and EL2 Val180, Cys181 and Ser182 of D3R may contribute to D3R specificity over D2R.

Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the ß2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of ß2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

N-linked Glycosylation on the N-terminus of the dopamine D2 and D3 receptors determines receptor association with specific microdomains in the plasma membrane.

Numerous G protein-coupled receptors (GPCRs) are glycosylated at extracellular regions. The regulatory roles of glycosylation on receptor function vary across receptor types. In this study, we used the dopamine D2and D3receptors as an experimental model to understand the underlying principles governing the functional roles of glycosylation. We used the pharmacological inhibitor, tunicamycin, to inhibit glycosylation, generated chimeric D2and D3receptors by swapping their respective N-termini, and produced the glycosylation site mutant D2and D3receptors to study the roles of glycosylation on receptor functions, including cell surface expression, signaling, and internalization through specific microdomains. Our results demonstrate that glycosylation on the N-terminus of the D3 receptor is involved in the development of desensitization and proper cell surface expression. In addition, glycosylation on the N-terminus mediates the internalization of D2and D3receptors within the caveolae and clathrin-coated pit microdomains of the plasma membrane, respectively, by regulating receptor interactions with caveolin-1 and clathrin. In conclusion, this study shows for the first time that glycosylation on the N-terminus of GPCRs is involved in endocytic pathway selection through specific microdomains. These data suggest that changes in the cellular environment that influence posttranslational modification could be an important determinant of intracellular GPCR trafficking.

Multiple signaling routes involved in the regulation of adenylyl cyclase and extracellular regulated kinase by dopamine D(2) and D(3) receptors.

Most G protein coupled receptors (GPCR) regulate multiple cellular processes by coupling to more than one kind of G protein. Furthermore, recent studies have reported G protein-independent/ß-arrestin-dependent signaling pathway for some GPCRs. Dopamine D(2) and D(3) receptors (D(2)R, D(3)R), the major targets of currently used antipsychotic drugs, are co-expressed in some of the same dopaminergic neurons and regulate the same overlapping effectors. However, the specific subunits of G proteins that regulate each signaling pathway are not clearly identified. In addition, the existence of ß-arrestin-dependent/G protein-independent signaling is not clear for these receptors. In this study, we determined the G protein subtypes and ß-arrestin dependency involved in the signaling of D(2)R and D(3)R, which was measured by inhibition of adenylyl cyclase and extracellular signal-regulated kinase (ERK) activation. For the inhibition of cAMP production in HEK-293 cells, D(2)R used the Gαo subunit but D(3)R used the ßγ subunit of Gi family proteins. For the regulation of ERK activation, D(2)R used the α subunits of Gi/o proteins both in HEK-293 cells and COS-7 cells, but D(3)R used Gαo and Gßγ in HEK-293 cells and COS-7 cells, respectively. ß-Arrestin-dependent/G protein-independent ERK activation was not observed for both D(2)R and D(3)R. Agonist-induced ß-arrestin translocation was observed with D(2)R but not with D(3)R, and ß-arrestins exerted inhibitory influences on G protein-dependent ERK activation by D(2)R, but not D(3)R. These results show that the D(2)R and D(3)R, which have overlapping cellular expressions and functional roles, employ distinct G protein subunits depending on the cell types and the effectors they control.

RGS4 exerts inhibitory activities on the signaling of dopamine D2 receptor and D3 receptor through the N-terminal region.

Dopamine D(2) receptor and D(3) receptor (D(2)R and D(3)R) are the major targets for current antipsychotic drugs, and their proper regulation has pathological and pharmacological significance. This study was conducted to understand the functional roles and molecular mechanisms of RGS proteins (RGS2, RGS4, and RGS9-2) on the signaling of D(2)R and D(3)R. RGS proteins were co-expressed with D(2)R and D(3)R in HEK-293 cells. The protein interactions between RGS proteins and D(2)R/D(3)R, and effects of RGS proteins on the internalization, signaling, and desensitization of D(2)R/D(3)R were determined. In addition, the RGS4 proteins were subdivided into N-terminal region, RGS domain, and the C-terminal region, and the specific subdomain of RGS4 protein involved in the regulation of the signaling of D(2)R/D(3)R was determined. All of RGS proteins we tested interacted with D(2)R/D(3)R. RGS4 exerted potent inhibitory activities on the signaling of D(2)R/D(3)R. RGS9-2 exerted selective but moderate inhibitory activity on D(3)R and the internalization of D(2)R. RGS2 had no effect. The N-terminal domain of RGS4 was involved in its interaction with D(2)R and D(3)R and was required for the inhibitory activity of the RGS domain. The study for the first time showed that RGS4 is the major RGS protein which interacts through the N-terminal region and exerts potent inhibitory activities on the signaling of D(2)R and D(3)R.

ß-arrestin2 plays permissive roles in the inhibitory activities of RGS9-2 on G protein-coupled receptors by maintaining RGS9-2 in the open conformation.

Together with G protein-coupled receptor (GPCR) kinases (GRKs) and ß-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D(3) receptor (D(3)R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, ß-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, Gß5, R7-binding protein (R7BP), and D(3)R. The DEP domain of RGS9-2, under the permission of ß-arrestin2, inhibited the signaling of D(3)R in collaboration with Gß5. ß-Arrestin2 competed with R7BP and Gß5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for ß-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that ß-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.

Novel regulatory mechanism of canonical Wnt signaling by dopamine D2 receptor through direct interaction with beta-catenin.

Classical G protein-coupled receptors (GPCRs) and canonical Wnt pathways were believed to use distinct signaling pathways. However, recent studies have shown that these two pathways interact each other by sharing several intermediate signaling components. Recent in vivo studies showed that antipsychotic drugs, which block dopamine D2-like receptors, increase the cellular levels of downstream signaling components of canonical Wnt pathways, such as dishevelled (Dvl), glycogen synthase kinase 3ß (GSK3ß), and ß-catenin. These results suggest that some functional interactions might exist between Wnt pathway and D2-like receptors. In this study, we show that among five different dopamine receptor subtypes, D(2) receptor (D(2)R) selectively inhibited the Wnt signaling, which was measured by lymphoid enhancing factor-1 (LEF-1)-dependent transcriptional activities. D(2)R-mediated inhibition of Wnt signaling was agonist- and G protein-independent and did not require receptor phosphorylation or endocytosis. D(2)R inhibited the LEF-1-dependent transcriptional activities, and this inhibitory activity was not affected by the inhibition of GSK-3ß, suggesting that D(2)R inhibited the Wnt signaling by acting on the downstream of GSK3ß. D(2)R directly interacted with ß-catenin through the second and third loops, leading to a reduction of ß-catenin distribution in the nucleus, resulting in an inhibition of LEF-1-dependent transcription. This is a novel mechanism for the regulation of canonical Wnt signaling by GPCRs, in which receptor proteins recruit ß-catenin from cytosol to the plasma membrane, resulting in the decrement of the ß-catenin/LEF-1-dependent transcription in the nucleus.

Agonist-induced endocytosis and receptor phosphorylation mediate resensitization of dopamine D(2) receptors.

The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.

Characterization of functional roles of DRY motif in the 2nd intracellular loop of dopamine D2 and D3 receptors.

Dopamine D(2)R and D(3)R (D(2)R, D(3)R) show very high sequence homology and employ virtually identical signaling pathways even though D(2)R is 2 approximately 5 times more active. Among the structural motifs identified, a triplet sequence, Asp-Arg-Tyr (DRY motif), plays critical roles in the determination of receptor conformations for signaling and intracellular trafficking of G protein-coupled receptors by forming intramolecular interactions. Thus, it is possible that different signaling efficiencies of D(2)R and D(3)R might be caused by the receptor activation levels stabilized by their own DRY motifs. In this study, the Arg and Asp residues of D(2)R and D(3)R were mutated, and resulting changes in their signaling and intracellular trafficking properties were comparatively studied. Mutation of the Arg residues of D(2)R and D(3)R abolished their signaling but differently affected their intracellular localizations. The wildtype and R132H-D(2)R were expressed mainly on the plasma membrane. On the other hand, compared with the wildtype D(3)R, a substantial amount of R128H-D(3)R was localized intracellularly. The expression of receptor proteins on the plasma membrane and their signaling efficiencies were more drastically affected by the mutation of the Asp residue of D(3)R than D(2)R. Therefore, it was concluded that the different levels of conformational strain exerted by the DRY motif might partly determine the quantitative differences in the signaling efficiencies between D(2)R and D(3)R.