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 ␤-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 (5, 6). The D 2 R and D 3 R, which are major potential targets for antipsychotic drugs, differ in pharmacological profiles and in brain distribution. D 3 R displays much higher affinity for endogenous dopamine, and its distribution in the brain is predominantly localized to the limbic area (7).The architectures of the D 2 R and D 3 R are similar, with the two sharing 46% overall amino acid homology and 78% identity in the transmembrane domains (8). Similarly D 2 R and D 3 R share many signaling properties when they are expressed in mammalian cells. For example, both regulate adenylyl cyclase (9 -11), extracellular acidification (Na ϩ /H ϩ exchange) (9, 12, 13), mitogenesis (9, 14), mitogen-activated protein kinase activation (15, 16), dopamine release (17), and ion channel function (18,19). Although it was reported that D 3 R is exclusively expressed in specific limbic areas such as the islands of Calleja and nucleus accumbens (7), more recent studies in monkey and human brain have shown that D 3 R is also expressed in mesencephalic dopaminergic neurons (20) and seems to be coexpressed with D 2 R in the same cells (21). The question arises why two structurally and functionally similar receptor proteins are expressed in both the same and distinct regions of the brain. Unless one of these receptors utilizes an uncharacterized and unshared signaling pathway, one speculation would be that different brain regions require different regulatory properties of the receptors.A common paradigm of G protein-coupled receptor desensitization is that agonist-induced receptor signaling is rapidly attenuated via G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and arrestin binding (22). The arrestin family of proteins to which the ␤-arrestins belong initiates receptor internalization through clathrin-coated pits (sequestration) (23,24), and in this process additional components of the endocytic machinery like dynamin and ␤ 2 -adaptin are known to be involved (25)(26)(27)(28). The sequestration of the D 2 R

Extracellular signal-regulated kinase 1 (ERK1) 1 and ERK2 (p44 ERK and p42 ERK ) are key cellular components that control cell proliferation and differentiation (1). The regulation of ERK through G protein-coupled receptors (GPCRs) is a complicated process. Various signaling components play different roles in ERK activation depending on the GPCR and cell types involved (2).There has been substantial progress in the understanding of cellular events that link the activation of GPCR and ERK (for review, see Ref. 1). These signaling events can be classified into several distinct pathways. They include: (i) Ras-dependent activation of ERK via transactivation of receptor-tyrosine kinases (RTKs) such as EGFR, (ii) Ras-independent ERK activation via protein kinase C (PKC) that converges with RTK signaling at the Raf level (iii) activation or inhibition of ERK via the cAMP/ protein kinase A (PKA) pathway, in which the direction of regulation depends on the type of Raf involved, and (iv) the recently substantiated ␤-arrestin-mediated pathway proven in certain classes of GPCRs (3). However, it should be mentioned that these signaling pathways are deduced from the limited sets of individual receptors or cell types, and more extensive and systemic studies are needed for these signaling models to be generalized (4). Among all the dopamine receptor subtypes characterized, it is generally accepted that D 2 R and D 3 R are related to schizophrenia. Possibly because of high similarity in their amino acid composition (46% overall amino acid homology and 78% identity in the transmembrane domains) (5), D 2 R and D 3 R share most signaling pathways such as adenylyl cyclase, extracellular acidification, mitogenesis, ERK activation, inhibition of dopamine synthesis, and ion channel regulation (K ϩ , Ca 2ϩ ) (for review, see Ref. 6). Furthermore, recent studies show that although D 3 R is more densely expressed in the limbic area, mesencephalic dopaminergic neurons express both D 2 R and D 3 R (7-9). Because D 2 R and D 3 R are virtually the same in functional aspect and are expressed in the same cells, it can be speculated that signaling routes or regulatory mechanisms for functions of effectors could be different. To address this issue, we decided to focus on a specific cellular function and conduct a detailed study on signaling mechanisms in order to see if their signaling or regulatory pathways differ. ERK activation was selected as a model experimental system, because GPCRmediated ERK regulation consists of multiple complex steps, which are relatively well established. D 2 R-mediated ERK activation has been reported from different cell types. It is pertussis toxin (PTX)-sensitive and partially blocked by the dominant negative mutant of Ras, N17Ras, in

No abstract

Homologous desensitization of D 1 dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D 1 receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.

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 β2AR-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 β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

The structure activity relationship of flavonoids for anti-allergic actions was studied by determining the IC50 values for the degranulation. The hexosaminidase release from RBL-2H3 cells (degranulation marker) was employed as an estimate for the anti-allergic actions. Among 22 flavonoid compounds tested, luteolin, apigenin, diosmetin, fisetin, and quercetin were found to be most active with IC50 values less than 10 microM.

Although the corazonin gene (Crz) has been molecularly characterized, little is known concerning the function of this neuropeptide in Drosophila melanogaster. To gain insight into Crz function in Drosophila, we have investigated the developmental regulation of Crz expression and the morphology of corazonergic neurons. From late embryo to larva, Crz expression is consistently detected in three neuronal groups: dorso-lateral Crz neurons (DL), dorso-medial Crz neurons (DM), and Crz neurons in the ventral nerve cord (vCrz). Both the vCrz and DM groups die via programmed cell death during metamorphosis, whereas the DL neurons persist to adulthood. In adults, Crz is expressed in a cluster of six to eight neurons per lobe in the pars lateralis (DLP), in numerous neuronal cells in the optic lobes, and in a novel group of four abdominal ganglionic neurons present only in males (ms-aCrz). The DLP group consists of two subsets of cells having different developmental origins: embryo and pupa. In the optic lobes, we have detected both Crz transcripts and Crz promoter activity, but no Crz-immunoreactive products, suggesting a post-transcriptional regulation of Crz mRNA. Projections of the ms-aCrz neurons terminate within the ventral nerve cord, implying a role as interneurons. Terminals of the DLP neurons are found in the retrocerebral complex that produces juvenile hormone and adipokinetic hormone. Significant reduction of trehalose levels in adults lacking DLP neurons suggests that DLP neurons are involved in the regulation of trehalose metabolism. Thus, the tissue-, stage-, and sex-specific expression of Crz and the association of Crz with the function of the retrocerebral complex suggest diverse roles for this neuropeptide in Drosophila.

Among the characterized dopamine receptor subtypes, D₂ receptor (D₂R) and D₃ receptor (D₃R) are the main targets of neuroleptics that are currently in use. In particular, D₃R is closely related to the etiology of schizophrenia and drug addiction. The spatial expression patterns of D₂R and D₃R are distinct in certain areas of the brain. D₂R are heavily expressed in the regions responsible for motor functions, whereas D₃R are more selectively expressed in the limbic regions, which are associated with cognitive and emotional functions. Therefore, disturbances in the motor and endocrine functions, which are the most serious problems caused by the current neuroleptics, are likely to result from the non-selective blockade of D₂R. Selective regulation of D₃R is needed to separate the desired therapeutic activities from unwanted side effects that result from promiscuous blockade of other receptors. D₂R and D₃R possess high sequence homology and employ similar signaling pathways, and it is difficult to selectively regulate them. In this review, we discuss the signaling mechanisms, intracellular trafficking, and desensitization properties of D₂R and D₃R. In addition, the proteins interacting with D₂R or D₃R are discussed in relation to their roles in the regulation of receptor functions, followed by the current status of the development of selective D₃R ligands.