Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Asymptomatic high flow subclavian steal in a patient with hemodialysis access.

Subclavian steal phenomenon due to proximal subclavian artery stenosis or occlusion is not un-common but often remains asymptomatic. We describe the case of a 66-year-old man with end-stage renal disease hemodialysed through a brachio-brachial loop graft of the left forearm. Echo-Doppler precerebral examination showed a high reversed flow of 570 ml/min in the ipsilateral vertebral artery. After successful endovascular recanalization of the subclavian artery, access blood flow increased and vertebral flow decreased to 30 ml/min. Complete neurological examination was normal both before and after endovascular treatment. This case demonstrates how high a subclavian steal can be without causing symptoms and how well precerbral and cerebral circulation can adapt to hemodynamic changes.

Verification at the protein level of the PIF4-mediated external coincidence model for the temperature-adaptive photoperiodic control of plant growth in Arabidopsis thaliana.

Plant circadian clock controls a wide variety of physiological and developmental events, which include the short-days (SDs)-specific promotion of the elongation of hypocotyls during de-etiolation and also the elongation of petioles during vegetative growth. In A. thaliana, the PIF4 gene encoding a phytochrome-interacting basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this photoperiodic control of plant growth. According to the proposed external coincidence model, the PIF4 gene is transcribed precociously at the end of night specifically in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by the light-activated phyB and also the residual proteins are inactivated by the DELLA family of proteins. A number of previous reports provided solid evidence to support this coincidence model mainly at the transcriptional level of the PIF 4 and PIF4-traget genes. Nevertheless, the diurnal oscillation profiles of PIF4 proteins, which were postulated to be dependent on photoperiod and ambient temperature, have not yet been demonstrated. Here we present such crucial evidence on PIF4 protein level to further support the external coincidence model underlying the temperature-adaptive photoperiodic control of plant growth in A. thaliana.

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors

Plant growth and development are particularly sensitive to changes in the light environment and especially to vegetational shading. The shade-avoidance response is mainly controlled by the phytochrome photoreceptors. In Arabidopsis, recent studies have identified several related bHLH class transcription factors (PIF, for phytochrome-interacting factors) as important components in phytochrome signaling. In addition to a related bHLH domain, most of the PIFs contain an active phytochrome binding (APB) domain that mediates their interaction with light-activated phytochrome B (phyB). Here we show that PIF4 and PIF5 act early in the phytochrome signaling pathways to promote the shade-avoidance response. PIF4 and PIF5 accumulate to high levels in the dark, are selectively degraded in response to red light, and remain at high levels under shade-mimicking conditions. Degradation of these transcription factors is preceded by phosphorylation, requires the APB domain and is sensitive to inhibitors of the proteasome, suggesting that PIF4 and PIF5 are degraded upon interaction with light-activated phyB. Our data suggest that, in dense vegetation, which is rich in far-red light, shade avoidance is triggered, at least partially, as a consequence of reduced phytochrome-mediated degradation of transcription factors such as PIF4 and PIF5. Consistent with this idea, the constitutive shade-avoidance phenotype of phyB mutants partially reverts in the absence of PIF4 and PIF5

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism.

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle's loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl--dependent HCO3- secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1-/- mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1-/- mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO3- secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.

A dynamic model for stem cell homeostasis and patterning in Arabidopsis meristems.

Plants maintain stem cells in their meristems as a source for new undifferentiated cells throughout their life. Meristems are small groups of cells that provide the microenvironment that allows stem cells to prosper. Homeostasis of a stem cell domain within a growing meristem is achieved by signalling between stem cells and surrounding cells. We have here simulated the origin and maintenance of a defined stem cell domain at the tip of Arabidopsis shoot meristems, based on the assumption that meristems are self-organizing systems. The model comprises two coupled feedback regulated genetic systems that control stem cell behaviour. Using a minimal set of spatial parameters, the mathematical model allows to predict the generation, shape and size of the stem cell domain, and the underlying organizing centre. We use the model to explore the parameter space that allows stem cell maintenance, and to simulate the consequences of mutations, gene misexpression and cell ablations.

Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acide-Sensing Ion Channels)

RESUME: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse Les canaux sodiques ASICs (Acid-Sensing Ion Channels) participent à la signalisation neuronale dans les systèmes nerveux périphérique et central. Ces canaux non voltage dépendants sont impliqués dans l'apprentissage, l'expression de la peur, la neurodégénération consécutive à une attaque cérébrale et la douleur. Les bases moléculaires sous-tendant leur activité ne sont pas encore totalement comprises. Ces canaux sont activés par une acidification du milieu extracellulaire et régulés, entre autres, par des ions tels que le Ca2+, le Zn2+ et le CI". La cristallisation de ASIC inactivé a été publiée. Le canal est un trimére de sous-unités identiques ou homologues. Chaque sous-unité a été décrite en analogie à un avant bras, un poignet et une main constituée d'un pouce, d'un doigt, d'une articulation, une boule β et une paume. Nous avons appliqué une approche bioinformatique systématique pour identifier les pH senseurs putatifs de ASICIa. Le rôle des pH senseurs putatifs a été testé par mutagénèse dirigée et des modifications chimiques combinées à une analyse fonctionnelle afin de comprendre comment les variations de ρ H ouvrent ces canaux. Les pH senseurs sont des acides aspartiques et glutamiques éparpillés sur la boucle extracellulaire suggérant que les changements de pH contrôlent l'activation et l'inactivation de ASIC en (dé)protonant ces résidus en divers endroits de la protéine. Par exemple lors de l'activation, la protonation des résidus à l'interface entre le pouce, la boule β et le doigt d'une même sous-unité induit un mouvement du pouce vers la bouie β et le doigt. De même lors de l'inactivation du canal les paumes des trois sous-unités formant une cavité se rapprochent. D'après notre approche bioinformatique, aucune histidine n'est impliquée dans la détection des variations de pH extracellulaire c'est-à-dire qu'aucune histidine ne serait un pH-senseur. Deux histidines de ASIC2a lient le Zn2+ et modifient l'affinité apparente du canal pour les protons. Une seule des deux est conservée parmi tous les ASICs, hASICIa H163. Elle forme un réseau de liaison hydrogène avec ses voisins conservés. L'étude détaillée de ce domaine, Pinterzone, montre son importance dans l'expression fonctionnelle des canaux. La perturbation de ce réseau par l'introduction d'un résidu hydrophobe (cystéine) par mutagénèse dirigée diminue l'expression du canal à la membrane plasmique. La modification des cystéines introduites par des réactifs spécifiques aux groupements sulfhydryle inhibe les canaux mutés en diminuant leur probabilité d'ouverture. Ces travaux décrivent les effets de l'acidification du milieu extracellulaire sur les canaux ASICs. ABSTRACT: Study of pH-dependent activation and inactivation of ASIC channels Benoîte BARGETON, Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1G05 Lausanne, Switzerland The ASIC (Acid-Sensing Ion Channels) sodium channels are involved in neuronal signaling in the central and peripheral nervous system. These non-voltage-gated channels are involved in learning, the expression of fear, neurodegeneration after ischemia and pain sensation. The molecular bases underlying their activity are not yet fully understood. ASICs are activated by extracellular acidification and regulated, eg by ions such as Ca2+, the Zn2+ and CI". The crystallization of inactivated ASIC has been published. The channel is a trimer of identical or homologous subunits. Each subunit has been described in analogy to a forearm, wrist and hand consisting of a thumb, a finger, a knuckle, a β-ball and a palm. We applied a systematic computational approach to identify putative pH sensor(s) of ASICIa. The role of putative pH sensors has been tested by site-directed mutagenesis and chemical modification combined with functional analysis in order to understand how changes in pH open these channels. The pH sensors are aspartic and glutamic acids distributed throughout the extracellular loop, suggesting that changes in pH control activation and inactivation of ASIC by protonation / deprotonation of many residues in different parts of the protein. During activation the protonation of various residues at the interface between the finger, the thumb and the β-ball induces the movement of the thumb toward the finger and the β-ball. During inactivation of the channel the palms of the three subunits forming a cavity approach each other. No histidine has been shown to be involved in extracellular pH changes detection, i.e. no histidine is a pH- sensor. Two histidines of ASIC2 bind Zn2+ and alter the apparent affinity of channel for protons. Only one of the two His is conserved among all ASICs, hASICIa H163. This residue is part of a network of hydrogen bonding with its conserved neighbors. The detailed study of this area, the interzone, shows its importance in the functional expression of ASICs. Disturbance of this network by the introduction of hydrophobic residues decreases the cell surface channel expression. Chemical modification of the introduced cysteines by thiol reactive compounds inhibits the mutated channels by a reduction of their open probability. These studies describe the effects of extracellular acidification on ASICs. RESUME GRAND PUBLIC: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse La transmission synaptique est un processus chimique entre deux neurones impliquant des neurotransmetteurs et leurs récepteurs. Un dysfonctionnement de certains types de synapses est à l'origine de beaucoup de troubles nerveux, tels que certaine forme d'épilepsie et de l'attention. Les récepteurs des neurotransmetteurs sont de très bonnes cibles thérapeutiques dans de nombreuses neuropathologies. Les canaux ASICs sont impliqués dans la neurodégénération consécutive à une attaque cérébrale et les bloquer pourraient permettre aux patients d'avoir moins de séquelles. Les canaux ASICs sont des détecteurs de l'acidité qui apparaît lors de situations pathologiques comme l'ischémie et l'inflammation. Ces canaux sont également impliqués dans des douleurs. Cibler spécifiquement ces canaux permettrait d'avoir de nouveaux outils thérapeutiques car à l'heure actuelle l'inhibiteur de choix, l'amiloride, bloque beaucoup d'autres canaux empêchant son utilisation pour bloquer les ASICs. C'est pourquoi il faut connaître et comprendre les bases moléculaires du fonctionnement de ces récepteurs. Les ASICs formés de trois sous-unités détectent les variations de l'acidité puis s'ouvrent transitoirement pour laisser entrer des ions chargés positivement dans la cellule ce qui active la signalisation neuronale. Afin de comprendre les bases moléculaires de l'activité des ASICs nous avons déterminé les sites de liaison des protons (pH-senseurs), ligands naturels des ASICs et décrit une zone importante pour l'expression fonctionnelle de ces canaux. Grâce à une validation systématique de résultats obtenus en collaboration avec l'Institut Suisse de Bioinformatique, nous avons décrit les pH-senseurs de ASICIa. Ces résultats, combinés à ceux d'autres groupes de recherche, nous ont permis de mieux comprendre comment les ASICs sont ouverts par une acidification du milieu extracellulaire. Une seconde étude souligne le rôle structural crucial d'une région conservée parmi tous les canaux ASICs : y toucher c'est diminuer l'activité de la protéine. Ce domaine permet l'harmonisation des changements dus à l'acidification du milieu extracellulaire au sein d'une même sous-unité c'est-à-dire qu'elle participe à l'induction de l'inactivation due à l'activation du canal Cette étude décrit donc quelle région de la protéine atteindre pour la bloquer efficacement en faisant une cible thérapeutique de choix.

Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria.

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

AMADEUS: advanced manipulation for deep underwater sampling

AMADEUS is a dexterous subsea robot hand incorporating force and slip contact sensing, using fluid filled tentacles for fingers. Hydraulic pressure variations in each of three flexible tubes (bellows) in each finger create a bending moment, and consequent motion or increase in contact force during grasping. Such fingers have inherent passive compliance, no moving parts, and are naturally depth pressure-compensated, making them ideal for reliable use in the deep ocean. In addition to the mechanical design, development of the hand has also considered closed loop finger position and force control, coordinated finger motion for grasping, force and slip sensor development/signal processing, and reactive world modeling/planning for supervisory `blind grasping¿. Initially, the application focus is for marine science tasks, but broader roles in offshore oil and gas, salvage, and military use are foreseen. Phase I of the project is complete, with the construction of a first prototype. Phase I1 is now underway, to deploy the hand from an underwater robot arm, and carry out wet trials with users.