Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway

beta-arrestins are multifunctional proteins that act as scaffolds and transducers of intracellular signals from heptahelical transmembrane-spanning receptors (7TMR). Hedgehog (Hh) signaling, which uses the putative 7TMR, Smoothened, is established as a fundamental pathway in development, and unregulated Hh signaling is associated with certain malignancies. Here, we show that the functional knockdown of beta-arrestin 2 in zebrafish embryos recapitulates the many phenotypes of Hh pathway mutants. Expression of wild-type beta-arrestin 2, or constitutive activation of the Hh pathway downstream of Smoothened, rescues the phenotypes caused by beta-arrestin 2 deficiency. These results suggest that a functional interaction between beta-arrestin 2 and Smoothened may be critical to regulate Hh signaling in zebrafish development.     

Targeting of diacylglycerol degradation to M1 muscarinic receptors by beta-arrestins

Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide-binding protein Gq and is terminated by receptor desensitization and degradation of the second messengers. We show that beta-arrestins coordinate both processes for the Gq-coupled M1 muscarinic receptor. beta-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, beta-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the beta-arrestin-DGK complex to activated 7TMRs. The dual function of beta-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3',5'-monophosphate phosphodiesterases to Gs-coupled beta2-adrenergic receptors. Thus, beta-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.     

Patched1 regulates hedgehog signaling at the primary cilium

Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. We investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localized to cilia and inhibited Smoothened (Smo) by preventing its accumulation within cilia. When Shh bound to Ptc1, Ptc1 left the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function.     

Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.     

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-beta receptor (TbetaRII), which is itself a kinase, rather than by a GRK. Association with betaarr2 led to internalization of both receptors and down-regulation of TGF-beta signaling. Thus, the regulatory actions of beta-arrestins are broader than previously appreciated, extending to the TGF-beta receptor family as well.     

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.     

草莓果实中七次跨膜受体蛋白生物信息学及时空表达特征分析

以草莓为试材,基于拟南芥(Arabidopsis thaliana)、人(Homo sapiens)和家鼠(Mus musculus)等数据库信息,采用同源比对方法,结合ClustalX 2.0序列比对及MEGA 5.0系统进化树分析,对草莓基因组中7TMR的基础生物信息进行了分析。结果表明,草莓基因组中含有84个7TMR,包括23个亚家族。半定量RT-PCR分析表明,该家族全部成员中有33个成员随果实发育表达量明显上调,17个成员表达量明显下调;Real-time PCR分析进一步确认,部分7TMR成员的基因表达与果实发育及成熟过程的整个进程有着密切的关系。此外还发现,草莓中7TMR家族中含有一类特殊的亚家族蛋白,该亚家族与人和家鼠中的脂联素受体高度同源,表明草莓中7TMR存在着非G-蛋白偶联信号转导途径。草莓中类似脂联素受体亚家族ADIPOR包含5个成员,对这5个成员的时空表达和刺激应答进行了分析,发现其中某些成员不仅与果实发育进程关系密切,且对糖信号应答,暗示着该类蛋白在草莓果实发育和成熟调控中可能起着重要作用。     

The primary cilium as the cell's antenna: signaling at a sensory organelle

Almost every vertebrate cell has a specialized cell surface projection called a primary cilium. Although these structures were first described more than a century ago, the full scope of their functions remains poorly understood. Here, we review emerging evidence that in addition to their well-established roles in sight, smell, and mechanosensation, primary cilia are key participants in intercellular signaling. This new appreciation of primary cilia as cellular antennae that sense a wide variety of signals could help explain why ciliary defects underlie such a wide range of human disorders, including retinal degeneration, polycystic kidney disease, Bardet-Biedl syndrome, and neural tube defects.     

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

Enhanced morphine analgesia in mice lacking beta-arrestin 2

The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.     

Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins

Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane.     

Progression of vertebrate limb development through SHH-mediated counteraction of GLI3

Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.     

鳜嗅板的组织学研究

应用光学显微镜、扫描及透射电镜观察了鳜嗅板的组织结构。结果表明：鳜的嗅板不发达。初级嗅板排列方式属于Ｇ型。感觉上皮连续地分布在初级嗅板的外周区。由纤毛细胞、感觉细胞、支持细胞、粘液细胞及基底细胞组成。在扫描电镜下,纤毛细胞顶端的纤毛多而长;感觉细胞可分成有４￣８根短管状纤毛的纤毛感觉细胞和有微绒毛样突起的微绒毛感觉细胞;粘液细胞呈球形或棒状;支持细胞表面有微嵴。在航向电镜下,这些细胞具有一般细胞所     

鸡第四脑室底壁和侧壁扫描电镜结构观察

本实验用扫描电镜对8只产蛋高峰期母鸡第四脑室底壁和侧壁进行了观察,结果表明,鸡第四脑室布满草丛状纤毛和毡样微缄毛,纤毛的分布在不同区域有一定差别.室底壁正中沟纤毛最为稀疏,在其两侧各有一条纤毛密集带,由密集带向外,纤毛密度逐渐减小;侧壁纤毛较底壁稀疏,侧壁后部的纤毛比前部更密.室管膜细胞可分为多纤毛细胞和少纤毛细胞,前者主要分布于底壁的两侧和侧壁的中后部,后者主要分布于底壁的正中沟和侧壁的前部.鸡第四脑室中存在着神经元样细胞、神经胶质样细胞和类组织细胞,各类细胞见于该脑室的各部,还发现有独立的神经纤维穿行于第四脑室壁的表面.     

A conserved family of enzymes that phosphorylate inositol hexakisphosphate

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.     

Transduction of receptor signals by beta-arrestins

The transmission of extracellular signals to the interior of the cell is a function of plasma membrane receptors, of which the seven transmembrane receptor family is by far the largest and most versatile. Classically, these receptors stimulate heterotrimeric G proteins, which control rates of generation of diffusible second messengers and entry of ions at the plasma membrane. Recent evidence, however, indicates another previously unappreciated strategy used by the receptors to regulate intracellular signaling pathways. They direct the recruitment, activation, and scaffolding of cytoplasmic signaling complexes via two multifunctional adaptor and transducer molecules, beta-arrestins 1 and 2. This mechanism regulates aspects of cell motility, chemotaxis, apoptosis, and likely other cellular functions through a rapidly expanding list of signaling pathways.     

beta-Arrestin: a protein that regulates beta-adrenergic receptor function

Homologous or agonist-specific desensitization of beta-adrenergic receptors is thought to be mediated by a specific kinase, the beta-adrenergic receptor kinase (beta ARK). However, recent data suggest that a cofactor is required for this kinase to inhibit receptor function. The complementary DNA for such a cofactor was cloned and found to encode a 418-amino acid protein homologous to the retinal protein arrestin. The protein, termed beta-arrestin, was expressed and partially purified. It inhibited the signaling function of beta ARK-phosphorylated beta-adrenergic receptors by more than 75 percent, but not that of rhodopsin. It is proposed that beta-arrestin in concert with beta ARK effects homologous desensitization of beta-adrenergic receptors.