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

Zheng, M.,Zhang, X.,Sun, N.,Min, C.,Zhang, X.,Kim, K.M. Elsevier Biomedical Press 2016 Biochimica et biophysica acta, Molecular cell rese Vol.1863 No.8

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D<SUB>2</SUB> and D<SUB>3</SUB> receptors (D<SUB>2</SUB>R, D<SUB>3</SUB>R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D<SUB>2</SUB>R and D<SUB>3</SUB>R, comparative studies were conducted on the signaling and intracellular trafficking of the D<SUB>2</SUB>R and D<SUB>3</SUB>R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D<SUB>2</SUB>R and D<SUB>3</SUB>R. 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 D<SUB>2</SUB>R and D<SUB>3</SUB>R, through GRK2 and β-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D<SUB>2</SUB>R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D<SUB>2</SUB>R. Knockdown of FLNA or coexpression of active RalA prevented D<SUB>3</SUB>R from coupling with G protein. The selective involvement of GRK2- and β-arrestins in the RalA-mediated cellular processes of the D<SUB>2</SUB>R and D<SUB>3</SUB>R 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 D<SUB>2</SUB>R and D<SUB>3</SUB>R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and β-arrestins.

Roles of Conserved Intracellular Sequences Regions for the Proper Expression of Dopamine D2 and D3 Receptors

Eun-Young Cho,Jae H Park,김경만 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.5

The dopamine D2 receptor and D3 receptor (D2R, D3R) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the D3R signals are 2~5 times less efficient than D2R. Previous studies have suggested that conformational constraints of D3R might be responsible for the poor coupling with the G protein. To test this hypothesis, point mutations were introduced into some of the conserved regions between D2R and D3R, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the 1st intracellular loop, SS motif in the 2nd intracellular loop, YxxL and TxxS/xS motifs in the 3rd intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the 3rd intracellular loop markedly decreased the level of D3R expression compared with D2R. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between D2R and D3R in the 1st intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between D2R and D3R. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the 1st loop of D3R are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D2R has a more flexible overall conformation that can accept mutated residues in the intracellular region than D3R, which might be partly responsible for the quantitative differences in the signaling efficiency between D2R and D3R.

Roles of Conserved Intracellular Sequences Regions for the Proper Expression of Dopamine $D_2$ and $D_3$ Receptors

Cho, Eun-Young,Park, Jae-H.,Kim, Kyeong-Man 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.5

The dopamine $D_2$ receptor and $D_3$ receptor ($D_2R$, $D_3R$) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the $D_3R$ signals are $2{\sim}5$ times less efficient than $D_2R$. Previous studies have suggested that conformational constraints of $D_3R$ might be responsible for the poor coupling with the G protein. To test this hypothesis, point mutations were introduced into some of the conserved regions between $D_2R$ and $D_3R$, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the $1^{st}$ intracellular loop, SS motif in the $2^{nd}$ intracellularloop, YxxL and TxxS/xS motifs in the $3^{rd}$ intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the $3^{rd}$ intracellular loop markedly decreased the level of $D_3R$ expression compared with $D_2R$. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between $D_2R$ and $D_3R$ in the $1^{st}$ intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between $D_2R$ and $D_3R$. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the $1^{st}$ loop of $D_3R$ are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D_2R$ has a more flexible overall conformation that can accept mutated residues in the intracellular region than $D_3R$, which might be partly responsible for the quantitative differences in the signaling efficiency between $D_2R$ and $D_3R$.

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D₃ receptor signaling

Sun, Ningning,Zhang, Xiaowei,Guo, Shuohan,Le, Hang Thi,Zhang, Xiaohan,Kim, Kyeong-Man Elsevier 2018 Biochimica et biophysica acta, Molecular cell rese Vol.1865 No.9

<P><B>Abstract</B></P> <P>The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D<SUB>3</SUB> receptor (D<SUB>3</SUB>R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D<SUB>3</SUB>R. <I>SRC</I> proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D<SUB>3</SUB>R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D<SUB>3</SUB>R, which can be extended to the crosstalk between other RTKs and GPCRs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Stimulation of EGFR results in the tyrosine phosphorylation of GRK2. </LI> <LI> Gβγ, Src, and endocytosis of EGFR are required for the tyrosine phosphorylation of GRK2. </LI> <LI> Tyrosine phosphorylated GRK2 binds to T142 of D<SUB>3</SUB>R. </LI> <LI> GRK2-bound D<SUB>3</SUB>R is uncoupled from G proteins to result in signaling inhibition. </LI> <LI> GRK2-bound D<SUB>3</SUB>R undergoes endocytosis and degradation through lysosomal pathway. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Current Perspectives on the Selective Regulation of Dopamine D2 and D3 Receptors

Dong Im Cho,김경만,Mei Zheng 대한약학회 2010 Archives of Pharmacal Research Vol.33 No.10

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.

Characterization of Functional Roles of DRY Motif in the $2^{nd}$ Intracellular Loop of Dopamine $D_2$ and $D_3$ Receptors

Kim, Ju-Heon,Cho, Eun-Young,Min, Cheng-Chun,Park, Jae-H,Kim, Kyeong-Man 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.4

Dopamine $D_2R$ and $D_3R$ $(D_2R,\;D_3R)$ show very high sequence homology and employ virtually identical signaling pathways even though $D_2R$ is $2{\sim}5$ 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 $D_2R$ and $D_3R$ might be caused by the receptor activation levels stabilized by their own DRY motifs. In this study, the Arg and Asp residues of $D_2R$ and $D_3R$ were mutated, and resulting changes in their signaling and intracellular trafficking properties were comparatively studied. Mutation of the Arg residues of $D_2R$ and $D_3R$ abolished their signaling but differently affected their intracellular localizations. The wildtype and $R132H-D_2R$ were expressed mainly on the plasma membrane. On the other hand, compared with the wildtype $D_3R$, a substantial amount of $R128H-D_3R$ 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 $D_3R$ than $D_2R$. 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 $D_2R$ and $D_3R$.

Characterization of Functional Roles of DRY Motif in the 2nd Intracellular Loop of Dopamine D2 and D3 Receptors

Ju-Heon Kim,Eun-Young Cho,Chengchun Min,Jae H. Park,김경만 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.4

Dopamine D2R and D3R (D2R, D3R) show very high sequence homology and employ virtually identical signaling pathways even though D2R is 2 ~ 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 D2R and D3R might be caused by the receptor activation levels stabilized by their own DRY motifs. In this study, the Arg and Asp residues of D2R and D3R were mutated, and resulting changes in their signaling and intracellular trafficking properties were comparatively studied. Mutation of the Arg residues of D2R and D3R abolished their signaling but differently affected their intracellular localizations. The wildtype and R132H-D2R were expressed mainly on the plasma membrane. On the other hand, compared with the wildtype D3R, a substantial amount of R128H-D3R 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 D3R than D2R. 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 D2R and D3R.

Simultaneous Measurement of Dopamine Transporters and D2/D3 Receptors in Drug Naïve Schizophrenic Patients

Yuan-Hwa Chou,Wen-Sheng Huang,Ju-Wei Hsu,Shin-Min Lee,An-Shoei Yang,Kai-Chun Yang 대한정신약물학회 2010 CLINICAL PSYCHOPHARMACOLOGY AND NEUROSCIENCE Vol.8 No.2

Objective: Previous single photon emission computed tomography (SPECT) studies have demonstrated that co-injection of radiotracers 99mTc-TRODAT and 123IBZM can assess both presynaptic, dopamine transporters (DATs), and postsynaptic, dopamine D2/D3 receptors, markers of dopaminergic system simultaneously. The aim of this study was to determine both DATs and dopamine D2/D3 receptors in drug naïve schizophrenic patients. Methods: Severn drug naïve schizophrenic patients and eleven age-matched healthy controls were recruited. Each subject received one SPECT measurement after intravenous co-administration of 99mTc-TRODAT and 123IBZM and one brain MRI scan. A ratio equilibrium model was applied to calculate the specific uptake ratio (SUR) which corresponds to the ratio of Bmax (receptor density) and KD (affinity). Results: The results showed that the SUR of DATs binding was not significantly different between healthy controls and drug naïve schizophrenic patients (2.92±0.62 vs. 2.65±0.27, p=0.052) and, however, the SUR of dopamine D2/D3 receptors binding was significantly lower in drug naïve schizophrenic patients than those in healthy controls (2.79±0.54 vs. 3.90±0.58, p=0.001). Additionally, there was a significant correlation between left and right binding for both DATs (r=0.80, p=0.003) and dopamine D2/D3 receptors (r=0.96, p=0.000) in healthy controls but not seen in drug naïve schizophrenic patients (r=0.54, p=0.20 for DATs, r=0.70, p=0.08 for dopamine D2/D3 receptors). Conclusion: This study provides evidences to support the idea that abnormalities of dopaminergic system in schizophrenia and further elucidates the disruption of inter-hemispheric interaction of postsynaptic and presynaptic markers.

Functional Regulation of Dopamine D₃ Receptor through Interaction with PICK1

Zheng, Mei,Zhang, Xiaohan,Min, Chengchun,Choi, Bo-Gil,Oh, In-Joon,Kim, Kyeong-Man The Korean Society of Applied Pharmacology 2016 Biomolecules & Therapeutics(구 응용약물학회지) Vol.24 No.5

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 $D_3$ receptors ($D_3R$), which act as autoreceptors to negatively regulate dopaminergic neurons. PICK1 colocalized with both dopamine $D_2$ receptor ($D_2R$) and $D_3R$ in clusters but exerted different functional influences on them. The cell surface expression, agonist affinity, endocytosis, and signaling of $D_2R$ were unaffected by the coexpression of PICK1. On the other hand, the surface expression and tolerance of $D_3R$ were inhibited by the coexpression of PICK1. These findings show that PICK1 exerts multiple effects on $D_3R$ functions.

Roles of Dopamine D₂ Receptor Subregions in Interactions with β-Arrestin2

Zhang, Xiaohan,Choi, Bo-Gil,Kim, Kyeong-Man The Korean Society of Applied Pharmacology 2016 Biomolecules & Therapeutics(구 응용약물학회지) Vol.24 No.5

${\beta}$-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of ${\beta}$-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with ${\beta}$-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with ${\beta}$-arrestin2. For this, we employed dopamine $D_2$ and $D_3$ receptors ($D_2R$ and $D_3R$, respectively), since they display distinct agonist-induced interactions with ${\beta}$-arrestins. Our results showed that the second and third intracellular loops of $D_2R$ are involved in the agonist-induced translocation of ${\beta}$-arrestins toward plasma membranes. In contrast, the N- and C-termini of $D_2R$ exerted negative effects on the basal interaction with ${\beta}$-arrestins.