Arrestin‐dependent but G‐protein coupled receptor kinase‐independent uncoupling of D2‐dopamine receptors

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G‐protein gated inwardly rectifying potassium channels (K_ir3) and directly compared the effects of co‐expression of G‐protein coupled receptor kinase (GRK) and arrestin on agonist‐dependent desensitization of the receptor response. We found, as described previously, that co‐expression of a GRK and an arrestin synergistically increased the rate of agonist‐dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin‐dependent GRK‐independent desensitization of D2R‐K_ir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin‐dependent GRK‐independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist‐dependent desensitization even after GRK co‐expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

     

Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon β2‐adrenergic receptor activation

The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the “second wave” of signaling from endosomes. Here, we set out to determine whether G_s signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin‐coated structures (CCSs) or caveolae upon activation by β₂‐adrenergic receptors (β₂AR). Activated β₂AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after β₂AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gα_s and Gγ₂ exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon β₂AR stimulation, no redistribution to CCSs, and weak activation‐independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated G_s signaling cascade members and confirm that β₂AR and arrestin 3 accumulate in CCSs, while Gα_s, Gγ₂ and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.     

GISP binding to TSG101 increases GABA receptor stability by down-regulating ESCRT-mediated lysosomal degradation

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABA(B) receptor subunit GABA(B1). Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA(B2) subunit, their co-expression in HEK293 cells increases levels of GABA(B2). GISP also inhibits TSG101-dependent GABA(B2) down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA(B2) degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.     

Lithium selectively increases neuronal differentiation of hippocampal neural progenitor cells both in vitro and in vivo

Lithium has been demonstrated to increase neurogenesis in the dentate gyrus of rodent hippocampus. The present study was undertaken to investigate the effects of lithium on the proliferation and differentiation of rat neural progenitor cells in hippocampus both in vitro and in vivo. Lithium chloride (1-3 mM) produced a significant increase in the number of bromodeoxyuridine (BrdU)-positive cells in high-density cultures, but did not increase clonal size in low-density cultures. Lithium chloride at 1 mM (within the therapeutic range) also increased the number of cells double-labeled with BrdU antibody and TuJ1 (a class III beta-tubulin antibody) in high-density cultures and the number of TuJ1-positive cells in a clone of low-density cultures, whereas it decreased the number of glial fibrillary acidic protein-positive cells in both cultures. These results suggest that lithium selectively increased differentiation of neuronal progenitors. These actions of lithium appeared to enhance a neuronal subtype, calbindin(D28k)-positive cells, and involved a phosphorylated extracellular signal-regulated kinase and phosphorylated cyclic AMP response element-binding protein-dependent pathway both in vitro and in vivo. These findings suggest that lithium in therapeutic amounts may elicit its beneficial effects via facilitation of neural progenitor differentiation toward a calbindin(D28k)-positive neuronal cell type.     

Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.     

在低氧预处理延迟心肌保护中钙网蛋白表达升高

本文分别在整体实验和细胞培养条件下研究钙网蛋白（calreticulin,CRT）在低氧预处理（hypoxic preconditioning,HPC）延迟心肌保护中的表达及其信号转导机制。（1）整体实验时Wistar大鼠随机分为3组：假手术（sham）组、仅结扎冠状动脉的心肌缺血（myocardial infarction,MI）组和HPC后再结扎冠状动脉的HPC＋MI组,分别于术后24h、14d和28d观察HPC对缺血后心功能和梗死区、危险区面积的影响;采用Western blot检测CRT表达以及p38丝裂素活化蛋白激酶（p38mitogenactivated protein kinase,p38MAPK）、应激活化蛋白激酶（stress-activated protein kinase,SAPK）活性。（2）原代培养Sprague—Dawley乳鼠心肌细胞,随机分为6组：低氧,复氧（hypoxia／reoxygenation,H／R）组、HPC组、HPC＋H,R组、p38MAPK抑制剂SB203580＋HPC＋H／R（SB＋HPC＋H／R）组、SAPK抑制剂SP600125＋HPC＋H瓜（SP＋HPC＋H／R）组和正常对照组;采用台盼蓝排斥实验、乳酸脱氢酶（1actate dehydrogenase,LDH）活性检测及流式细胞仪检测各组细胞损伤情况;采用Western blot检测CRT表达及p38MAPK、SAPK的磷酸化水平。主要结果如下：（1）整体动物实验结果表明,HPC改善缺血对心肌左室压力最大上升,下降速度（＋dp／dtmax）的抑制,限制心肌梗死面积;HPC后CRT表达呈动态变化：术后24h时HPC＋MI组CRT表达增高106％（P〈0．05vsMI组）,以危险区最为显著;14d至28d表达逐步降低。相关分析显示,术后24h时CRT表达量与心功能呈正相关（r=0．9867,P〈0．05）,与梗死面积呈负相关（r=-0．9709,P〈0．05）。（2）细胞培养实验结果表明,HPC可减轻H／R诱导的心肌细胞LDH漏出,增加心肌细胞存活率,降低细胞凋亡;单纯HPC可诱导CRT表达轻度增加（222％,P〈0．05vs对照组）,而损伤性H／R诱导CRT过表达（503％,P〈0．05vs对照组）,HPC可降低H／R诱导CRT表达升高的幅度;p38MAPK活性与HPC诱导的CRT表达呈正相关（r=0．9021,P〈0．05）,而SAPK活性与其呈负相关（r=-0．8211,P〈0．05）。由此得出结论：（1）整体实验中HPC可明显改善缺血后心脏的收缩与舒张功能,限制心肌梗死范围,促进危险区心肌恢复;心肌梗死早期,HPC诱导CRT表达上调,参与心肌保护;（2）细胞培养实验中HPC可诱导CRT适度表达,增强原代培养乳鼠心肌细胞对H／R损伤的抵抗力;p38MAPK可能介导HPC诱导的CRT表达,而SAPK激活可能不利于心肌保护。     

MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells

MicroRNAs （or miRNAs） are small non-coding RNAs （21-25 nucleotides） that are involved in a wide range of activities related to the development and differentiation of cells. Comparison of the miRNA expression profiles of mouse P19 embryonic carcinoma cells with those of differentiated neural stem cells showed that the expression level of 65 miRNAs changed （2-fold） after differentiation. MiR-124a was dramatically upregulated （more than 20-fold） while miRNAs of the miR-302 family and those in the miR-290-295 cluster were strongly down-regulated. Further analysis revealed that some important factors such as Oct4 and Sox2 appeared to be involved in the regulation of these miRNAs. These results may contribute to a better understanding of miRNA-regulated neural differentiation in early mouse embryos.     

Arrestin domain‐containing protein 3 recruits the NEDD4 E3 ligase to mediate ubiquitination of the β2‐adrenergic receptor

Prolonged stimulation of the β2‐adrenergic receptor (β2AR) leads to receptor ubiquitination and downregulation. Using a genome‐wide RNA interference screen, we identified arrestin domain‐containing 3 (ARRDC3) as a gene required for β2AR regulation. The ARRDC3 protein interacts with ubiquitin ligase neural precursor development downregulated protein 4 (NEDD4) through two conserved PPXY motifs and recruits NEDD4 to the activated receptor. The ARRDC3 protein also interacts and co‐localizes with activated β2AR. Knockdown of ARRDC3 expression abolishes the association between NEDD4 and β2AR. Furthermore, functional inactivation of ARRDC3, either through small interfering RNA (siRNA)‐mediated knockdown or overexpression of a mutant that does not interact with NEDD4, blocks receptor ubiquitination and degradation. Our results establish ARRDC3 as an essential adaptor for β2AR ubiquitination.     

Osteoblast calcium-sensing receptor has characteristics of ANF/7TM receptors

There is evidence for a functionally important extracellular calcium-sensing receptor in osteoblasts, but there is disagreement regarding its identity. Candidates are CASR and a putative novel calcium-sensing receptor, called Ob.CASR. To further characterize Ob.CASR and to distinguish it from CASR, we examined the extracellular cation-sensing response in MC3T3-E1 osteoblasts and in osteoblasts derived from CASR null mice. We found that extracellular cations activate ERK and serum response element (SRE)-luciferase reporter activity in osteoblasts lacking CASR. Amino acids, but not the calcimimetic NPS-R568, an allosteric modulator of CASR, also stimulate Ob.CASR-dependent SRE-luciferase activation in MC3T3-E1 osteoblasts. In addition, we found that the dominant negative Galphaq(305-359) construct inhibited cation-stimulated ERK activation, consistent with Ob.CASR coupling to Galphaq-dependent pathways. Ob.CASR is also a target for classical GPCR desensitization mechanisms, since beta-arrestins, which bind to and uncouple GRK phosphorylated GPCRs, attenuated cation-stimulated SRE-luciferase activity in CASR deficient osteoblasts. Finally, we found that Ob.CASR and CASR couple to SRE through distinct signaling pathways. Ob.CASR does not activate RhoA and C3 toxin fails to block Ob.CASR-induced SRE-luciferase activity. Mutational analysis of the serum response factor (SRF) and ternary complex factor (TCF) elements in SRE demonstrates that Ob.CASR predominantly activates TCF-dependent mechanisms, whereas CASR activates SRE-luciferase mainly through a RhoA and SRF-dependent mechanism. The ability of Ob.CASR to sense cations and amino acids and function like a G-protein coupled receptor suggests that it may belong to the family of receptors characterized by an evolutionarily conserved amino acid sensing motif (ANF) linked to an intramembranous 7 transmembrane loop region (7TM).     

Agonist-induced internalization of histamine H2 receptor and activation of extracellular signal-regulated kinases are dynamin-dependent

Histamine H2 receptor (H2R) is a member of G protein-coupled receptor family. Agonist stimulation of H2R results in several cellular events including activation of adenylate cyclase and phospholipase C, desensitization of the receptor, activation of extracellular signal-regulated kinases ERK1/2, and receptor endocytosis. In this study, we identified a GTPase dynamin as a binding partner of H2R. Dynamin could associate with H2R both in vitro and in vivo. Functional analyses using dominant-negative form of dynamin (K44E-dynamin) revealed that cAMP production and the following H2R desensitization are independent of dynamin. However, the agonist-induced H2R internalization was inhibited by co-expression of K44E-dynamin. Furthermore, activation of extracellular-signal regulated kinases ERK1/2 in response to dimaprit, an H2R agonist, was attenuated by K44E-dynamin. Although H2R with truncation of 51 amino acids at its carboxy-terminus did not internalize after agonist stimulation, it still activated ERK1/2, but the degree of this activation was less than that of the wild-type receptor. Finally, K44E dynamin did not affect ERK1/2 activation induced by internalization-deficient H2R. These results suggest that the agonist-induced H2R internalization and ERK1/2 activation are partially dynamin-dependent. Furthermore, ERK1/2 activation via H2R is likely dependent of the endocytotic process rather than dynamin itself.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

Specific expression pattern of a novel Otx2 splicing variant during neural differentiation

We cloned a new splicing variant of Otx2 gene, Otx2c. Otx2c lacks entire exon 4, most of the region encoding the homeodomain. More importantly, Otx2c harbors an early premature stop codon and bioinformatics analysis prefers it to be a non-protein coding RNA. In addition, this splicing variant is not simply a noise during mRNA processing, since it is mainly expressed in undifferentiated human embryonic stem cells but gradually decreased during differentiation. Therefore, we report here that a single pre-mRNA can generate both coding and non-coding RNAs through alternative splicing and this splicing activity is tightly regulated in different cell contexts.     

Differential cytoskeletal association of ErbB1 and ErbB2 during keratinocyte differentiation

ErbB1 and ErbB2 display differential subcellular localization in human skin and cultured keratinocytes. To determine whether ErbB1 and ErbB2 also differ in cytoskeletal binding properties, normal human keratinocytes grown under conditions favoring a basal or differentiated phenotype were repeatedly extracted in a non-ionic detergent buffer. In basaloid keratinocytes, cytoskeletal association of ErbB1 and ErbB2 was limited. ErbB1 ( approximately 5%) was tightly associated with the cytoskeleton, compared to <1% of ErbB2 (p=0.004). After EGF stimulation, activated ErbB1 and ERK associated with the cytoskeleton to a greater extent than did total ErbB1 and total ERK. Association of ErbB2 increased markedly in differentiated keratinocytes, whereas association of ErbB1 was similar in basaloid and differentiated cells. Cytoskeletal association of ErbB2 correlated with plasma membrane localization. These results suggest that ErbB1 and ErbB2 employ different mechanisms of plasma membrane targeting during keratinocyte differentiation, and that cytoskeletal association may facilitate the coupling of activated ErbB1 and ERK.