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

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