92 research outputs found

    The molecular basis of V2 vasopressin receptor-G Protein coupling selectivity

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    G­Protein­gekoppelte Rezeptoren (GPCRs) stellen eine der größten in der Natur vorkommenden Proteinfamilien dar (Watson and Arkinstall, 1994). GPCRs sind plasmamembranständige Proteine, die mit heterotrimären G­Proteinen interagieren und eine Vielzahl an Signaltransduktionswegen aktivieren. Trotz der strukturellen Vielfalt der an GPCRs angreifenden Liganden stimulieren die meisten GPCRs nur eine begrenzte Anzahl strukturell sehr ähnlicher G­Proteine (Hedin et al., 1993; Conklin and Bourne, 1993). Die Aufklärung der molekularen Mechanismen, die dieser Rezeptor/G­Protein­Kopplungsselektivität zugrunde liegen, ist von fundamentaler Wichtigkeit für das Verständnis zellulärer Signaltransduktion. Ausführliche Struktur­Funktionsanalysen verschiedener Neurotransmitter­ rezeptoren, einschließlich der Muskarinrezeptoren (Wess, 1996) und adrenergen Rezeptoren (Dohlman et al., 1991; Savarese and Fraser, 1992; Strader et al., 1994), haben einen beträchtlichen Beitrag zur Identifizierung der strukturellen Elemente, die für die G­Protein­Kopplungsselektivität dieser Rezeptorgruppe verantwortlich sind, geleistet. Im Gegensatz dazu ist bisher noch weitgehend ungeklärt, welche molekularen Mechanismen der Kopplungsselektivität von GPCRs, die durch Peptidliganden aktiviert werden, zugrunde liegen. Das Ziel dieser Arbeit war daher, molekulare Grundlagen der G­Protein­ Kopplungsselektivität von Peptid­GPCRs näher zu untersuchen und aufzuklären. Die Vasopressinrezeptorfamilie unterscheidet sich von nahezu allen anderen Peptid­GPCRs darin, daß die einzelnen Rezeptorsubtypen deutlich unterschiedliche G­Protein­ Kopplungspräferenzen aufweisen. Die V1a­ und V1b­Vasopressinrezeptoren stimulieren selektiv G­Proteine der Gq/11 ­Familie, was zur Aktivierung von Phospholipase­Cbeta-Isomeren führt. Im Gegensatz dazu koppelt der V2­Vasopressinrezeptor vornehmlich an das G­Protein G s , was in einem Anstieg an intrazellulärem cAMP resultiert. Daher stellen die Vasopressinrezeptorsubtypen ein attraktives Modellsystem zum Studium der Peptid­ GPCR­Rezeptordomänen, die für die selektive G­Protein­Aktivierung verantwortlich sind, dar. Als Modellsystem für diese Arbeit diente primär der V2­Vasopressinrezeptor. Molekulare Faktoren, die die Gs ­ Kopplungsselektivität des V2­ Vasopressinrezeptors bestimmen. Eine frühere Studie zeigte, daß die Gegenwart der V1a­Rezeptorsequenz in der zweiten intrazellulären (i2) Schleife notwendig war, um den Wildtyp V1a und V1a/V2­ Rezeptorchimären effizient an Gq/11 ­Proteine zu koppeln (Liu and Wess, 1996). Effiziente Interaktionen zwischen Wildtyp V2 oder V1a/V2­Rezeptorchimären und dem G­Protein G s waren hingegen hauptsächlich von V2­Rezeptorsequenzen in der dritten intrazellulären (i3) Schleife abhängig. Um die molekularen Grundlagen der Gs ­ Kopplungsselektivität des V2­Rezeptors näher zu untersuchen, wurden zunächst klassische Mutagenesetechniken (zielgerichtete Mutagenese'') angewandt. Definierte V2­Rezeptorsegmente (oder einzelne Aminosäuren) wurden in den V1a­Rezeptor transferiert, und die resultierenden Hybrid­Vasopressinrezeptoren wurden anschließend in funktionellen Studien auf ihre Fähigkeit, hormonabhängig intrazelluläre cAMP­ Konzentrationen zu steigern (G s ­vermittelt), getestet. Diese Strategie schien besonders geeignet, da die Aktivierung des V1a­Wildtyprezeptors nahezu keine Auswirkungen auf intrazelluläre cAMP­Spiegel hat. Wie bereits erwähnt, ist die effiziente Kopplung des V2­Rezeptors an das Gs ­ Protein vornehmlich von V2­Rezeptorsequenzen in der i3­Schleife abhängig (Liu and Wess, 1996). Eine V1a­Rezeptormutante, deren i3­Schleife durch die homologe V2­ Rezeptorsequenz ersetzt worden war, war in der Lage, effizient mit Gs zu interagieren. Die Fähigkeit dieser Rezeptormutante, Gs zu aktivieren, war jedoch im Vergleich zum V2­Wildtyprezeptor vermindert. Diese Beobachtung ließ die Vermutung zu, daß noch andere intrazelluläre V2­Rezeptordomänen zur optimalen Gs ­Kopplung notwendig sind. Daher wurde zunächst eine Reihe von V1a/V2­Rezeptorchimären erzeugt, die den Beitrag der zweiten (i2) und vierten intrazellulären (i4) Rezeptordomäne zur V2­ Rezeptor/G s ­Kopplungsselektivität klären sollten. Funktionelle Untersuchungen der resultierenden Hybrid­Rezeptormutanten in Säugetierzellen (COS­7) zeigten, daß ein kurzes Segment im N­terminalen Abschnitt der i4­Domäne einen deutlichen Beitrag zur V2­Rezeptor/G s ­Kopplungsselektivität leistet. Eine V1a­Rezeptormutante, welche in der i3­Schleife und dem N­terminalen Segment der i4­Domäne (Ni4) homologe V2­ Rezeptorsequenzen enthielt, zeigte ein funktionelles Profil (EC 50 und E max ), welches mit dem V2­Wildtyprezeptor nahezu deckungsgleich war. Anschließend wurden strukturelle Elemente innerhalb der i3­Schleife näher untersucht. Funktionelle Analysen zeigten, daß der N­terminale Abschnitt der i3­Schleife weitgehend das G­Protein­Kopplungsprofil des V2­Rezeptors bestimmt. Eine Reihe von V1a­Rezeptormutanten wurde erzeugt, in denen kurze Segmente des N­terminalen Bereichs der i3­Schleife mit der entsprechenden V2­Rezeptorsequenz ausgetauscht wurden. Funktionelle Untersuchungen ergaben, daß ein Aminosäurepaar (Gln225, Val226) und ­triplet (Phe229, Arg 230, Glu231) am Beginn der i3­Schleife des V2­ Rezeptors für die effiziente Aktivierung von Gs von entscheidender Bedeutung sind. Durch Punktmutationen in diesem Bereich wurden zwei polare Aminosäuren, Gln225 und Glu231, identifiziert, die für die effiziente V2­Rezeptor/G s ­Interaktion essentiell sind. Untersuchungen mit anderen GPCR­Klassen (Dohlman et al., 1991; Savarese and Fraser, 1992; Strader et al., 1994; Wess, 1996) haben ebenfalls gezeigt, daß dem N­ Terminus der i3­Schleife eine besondere Rolle im Rezeptor/G­Protein­Kopplungsprozeß zukommt. In diesen Studien wird berichtet, daß vornehmlich hydrophobe und ungeladene Aminosäuren Schlüsselrollen in der rezeptorvermittelten G­Protein­Aktivierung einnehmen. Die hier beschriebenen Untersuchungen hingegen ergaben, daß zwei polare/geladene Aminosäuren, Gln225 und Glu231, für die V2­Rezeptor/G s ­Kopplung von besonderer Wichtigkeit sind und zeigen daher, daß die Rezeptor/G­Protein­ Kopplungsselektivität nicht auf ausschließlich hydrophoben Wechselwirkungen beruht. Desweiteren konnte beobachtet werden, daß die Länge der i3­Schleife die Effizienz, mit der der V2­Rezeptor G­Proteine der Gs ­Klasse zu aktivieren vermag, beeinflußen kann. Die V1a­ und V2­Rezeptoren weisen unterschiedlich lange i3­ Schleifen auf (die i3­Schleife des V2­Rezeptors ist 13 Aminosäuren kürzer als die des V1a­Rezeptors). Eine V1a­Rezeptormutante, deren N­terminaler Abschnitt der i3­ Schleife durch homologe V2­Rezeptorsequenz ersetzt wurde, konnte deutlich effizienter mit Gs interagieren, wenn der mittlere Abschnitt der i3­Schleife um elf Aminosäuren verkürzt wurde. Gleichermaßen konnte die effiziente Kopplung bestimmter V1a/V2­Hybridrezeptoren an Gs durch Einfügen von elf Aminosäuren in den zentralen Bereich der i3­Schleife deutlich gehemmt werden. Diese Ergebnisse legen nahe, daß der zentrale Bereich der i3­Schleife die Rezeptor/G­Protein­Kopplungsselektivität beeinflussen kann, obgleich diese Rezeptordomäne vermutlich nicht direkt mit dem G­Protein interagiert. Es ist denkbar, daß die Länge der i3­Schleife den Zugang des G­Proteins zu funktionell wichtigen Rezeptordomänen, z.B. Aminosäuren im Bereich der fünften Transmembrandomäne (TM V) und der i3­Schleife, reguliert. Identifizierung einzelner Aminosäuresubstitutionen und Aminosäuredeletionen, die die G­Protein­Kopplungsselektivität des V2­Rezeptors beeinflussen: Einsatz von Hefeexpressionstechnologie und zufallsgerichteter Mutagenese (random mutagenesis'') Im zweiten Teil dieser Arbeit wurden Hefe­(Saccharomyces cerevisiae)­ Expressionstechnologien angewandt, um Struktur­Funktionsanalysen des V2­Rezeptors zu erleichtern und Beschränkungen klassischer Mutagenesetechniken zu überwinden. Der V2­Wildtyprezeptor und verschiedene G­Proteinchimären aus Hefe­ und Säugetier­Galpha­ Untereinheiten wurden in genetisch modifizierten Hefelinien, deren Zellwachstum von effizienter Rezeptor/G­Protein­Kopplung abhängig war, co­exprimiert. In diesem System aktiviert produktive Rezeptor/G­Protein­Kopplung den Hefe­MAP­Kinase/Pheromon­ Signaltransduktionsweg. Dies führt zur Transkription des FUS1­HIS3­Reportergens und somit zur Expression von His3­Protein, was den Histidin­auxotrophen (his3) Hefelinien ermöglicht, in histidinfreiem Medium zu wachsen (Pausch et al., 1998). Es konnte gezeigt werden, daß heterolog exprimierte V2­Rezeptoren weder mit der Hefe­G­Protein­ alpha­Untereinheit (Gpa1p) noch mit einem mutierten Gpa1­Protein, in dem die C­terminalen fünf Aminosäuren gegen homologe Galpha q ­Sequenz ausgetauscht worden waren (Gq5), effizient interagierten. Im Gegensatz dazu erwies sich die Interaktion zwischen dem V2­ Rezeptor und einem mutierten Gpa1­Protein, dessen C­terminale fünf Aminosäuren die homologe Galpha s ­Sequenz enthielten (Gs5), als hocheffizient. Diese Beobachtungen zeigten, daß der V2­Rezeptor im Hefesystem sein physiologisches Kopplungsprofil beibehielt. Zur weiteren Validierung des Hefeexpressionssystems wurden die G q/11 ­ gekoppelten M 1 ­, M 3 ­ und M 5 ­Muskarinrezeptoren und verschiedene mutierte Vasopressin­ und M 3 ­Muskarinrezeptoren mit veränderten funktionellen Eigenschaften heterolog in Hefe exprimiert. Funktionelle Analysen zeigten, daß die Wildtyprezeptoren und die verschiedenen Rezeptormutanten in Hefe und Säugetierzellen ähnliche Phänotypen aufwiesen. Um zu untersuchen, weshalb der V2­Rezeptor nicht effizient an G­Proteine der Gq/11 ­Familie koppelt, sollte der in Hefe exprimierte V2­Rezeptor zufallsgerichteter Mutagenese (random mutagenesis'') unterzogen und Mutanten mit veränderten G­ Protein­Kopplungeigenschaften isoliert werden. Im speziellen wurde die i2­Schleife untersucht, da eine frühere Studie gezeigt hatte, daß vornehmlich die i2­Schleife des V1a­ Rezeptors für die V1a­Rezeptor/G q/11 ­Kopplungsselektivität verantwortlich ist (Liu and Wess, 1996). Mittels zufallsgerichteter Mutagenesetechnik wurde in Hefe eine Bibliothek von V2­Rezeptormutanten erzeugt, deren i2­Schleife Mutationen mit einer Mutageneserate von ungefähr 10% (auf der Nukleotidebene) enthielt. Anschließend wurden in einem Selektionsverfahren (screen'') 30 000 V2­Rezeptormutanten auf ihre Fähigkeit, mit Gq5 zu interagieren, überprüft. Es konnten vier V2­Rezeptormutanten isoliert werden, welche effizient an Gq5 (jedoch nicht an Hefe­Gpa1p) koppelten. Funktionelle Untersuchungen mit diesen und anderen mittels zielgerichteter Mutagenese erzeugter V2­Rezeptormutanten zeigten, daß die Substitution einer einzigen Aminosäure (Met145) im zentralen Bereich der i2­Schleife beträchtliche Auswirkungen auf die Rezeptor/G­Protein­Kopplungsselektivität hatte. Die Fähigkeit des V2­Rezeptors, produktiv mit Gq5 zu interagieren, war von der Anwesenheit relativ großer, hydrophober Aminosäuren wie Leucin und Tryptophan abhängig. Austausch von Met145 mit kleinen Aminosäuren wie Glycin oder Alanin erlaubte dem V2­Rezeptor nicht, Gq5 zu aktivieren. Interessanterweise interagierten alle V2­Rezeptormutanten, die eine Met145­ Punktmutation aufwiesen, mit Gs5 ähnlich effizient wie der V2­Wildtyprezeptor. Die Unfähigkeit der V2(Met145Gly)­ und V2(Met145Ala)­Rezeptoren, Gq5 zu aktivieren, beruht daher nicht auf einem Faltungsdefizit. Gleichermaßen basierte die Fähigkeit der V2(Met145Trp)­ und V2(Met145Leu)­Rezeptoren, produktiv an Gq5 zu koppeln, nicht auf der Überexpression von Rezeptorprotein. Diese Ergebnisse zeigen, daß die chemische Eigenschaft der Aminosäure an Position 145 die V2­Rezeptor/G­Protein­ Kopplungsselektivität reguliert. Interessanterweise befindet sich in allen anderen Subtypen der Vasopressin/Oxytocin­Rezeptorfamilie (V1a­, V1b­, und Oxytocin­ Rezeptoren), welche selektiv an G­Proteine der G q/11 ­Klasse gekoppelt sind, ein Leucin an der Stelle, die zu Met145 (V2­Rezeptorsequenz) homolog ist. Eine der vier ursprünglich isolierten V2­Rezeptormutanten enthielt neben verschiedenen Punktmutationen eine Deletion in Position Met145. In detaillierteren zielgerichteten Mutagenese­Studien wurden zwei V2­Rezeptormutanten erzeugt, die alle drei G­Proteine (Gq5, Gs5 und Gpa1p) aktivieren konnten. Um zu untersuchen, ob ein generelles Verkürzen der i2­Schleife um eine Aminosäure der Grund für die beobachtete Rezeptor/G­Protein­Promiskuität ist, wurden verschiedene V2­Rezeptormutanten erzeugt, in denen einzelne Aminosäuren unmittelbar N­ und C­terminal von Met145 deletiert worden waren. Funktionelle Untersuchungen ergaben, daß die Deletion einzelner Aminosäuren N­terminal von Met145 (Ile141delta, Cys142delta, Arg143delta oder Pro144delta) in V2­Rezeptormutanten resultierte, die nicht mit G­Proteinen interagieren konnten. Radioligand­Bindungsstudien zeigten, daß diese V2­Rezeptormutanten keine V2­Liganden binden konnten, was darauf schließen läßt, daß Deletionen einzelner Aminosäuren N­terminal von Met145 zu mißgefalteten Rezeptoren führen. Die Aminosäuren Ile141­Pro144 befinden sich am Beginn der i2­Schleife, unmittelbar neben der alpha­helikalen zytoplasmatischen Verlängerung der dritten Transmembrandomäne (TM III) in der Nähe des hochkonservierten DRY(H)­Motivs. Es ist denkbar, daß Aminosäuren innerhalb des Ile141­Pro144­Segments mit den zytoplasmatischen Abschnitten von TM III und/oder TM V interagieren und diese Wechselwirkungen die Rezeptorstruktur stabilisieren. Im Gegensatz dazu hatten Deletionen unmittelbar C­ terminal von Met145 (Leu146delta, Ala147delta, Tyr148delta oder Arg149delta) keinerlei Auswirkungen auf die Funktion des V2­Rezeptors. Diese Aminosäuren befinden sich im zentralen Bereich der i2­Schleife, der nicht mit den transmembranären Domänen des Rezeptorproteins interagieren kann

    The structure and function of the human ghrelin receptor

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    The peptide hormone, ghrelin, exerts its physiological effects through a G-protein-coupled receptor called the ghrelin-R. The ghrelin-R displays a high degree of constitutive activity, signalling through the inositol phosphate pathway in the absence of bound agonist. TMs III and VI have been reported to be central to the activation of Family A GPCRs, with interactions between the two helices stabilising the ground state. During activation conformational rearrangements result in these interactions being broken, with new contacts forming and stabilising the active state. Investigation of the ghrelin-R constitutive activity gives an insight into the mechanisms involved in receptor activation. In this study the role of specific individual residues in the ghrelin-R has been investigated and the effect of disrupting or introducing intramolecular interactions was addressed. Site-directed mutagenesis and functional assays revealed that ghrelin-R constitutive activity can be increased and decreased with mutation of residues within the TM domains, specifically TMs III, VI and VII. The extracellular loops have been found to be involved in ligand binding and activation in a number of Family A GPCRs. The residues within ECL2 of the ghrelin-R were systematically mutated to alanine to determine their role. In particular, one residue, Asn196, was identified as being critical in ghrelin-R function and may be forming stabilising interactions which maintain ghrelin-R constitutive activity. The data presented in this thesis provide an insight into the structure and function of the ghrelin-R and the underlying molecular mechanisms of ghrelin-R constitutive activity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

    A systems approach to determine how Toxoplasma gondii Infection causes neuropsychiatric disease

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    This thesis was previously held under moratorium from 16/03/2020 to 16/03/2022T. gondii infection acquired during life has been associated with psychoneurological disease in humans and behavioural changes in mice. However, less is known about the potential of congenitally acquired T. gondii infection, or for maternal T. gondii infection induced immune activation, to cause psychoneurological disease. The studies described herein, using LCMS (Liquid chromatography–mass spectrometry) demonstrate that adult acquired infection alters the neurochemistry and transcriptome of the brains of BALB/c mice. Notable changes to tryptophan, purine, arginine and carnitine metabolism were observed in infected mice. Congenitally infected and mice exposed to the maternal immune response to T. gondii, but not congenitally infected were found to have decreased mobility compared with control mice. Congenital T. gondii infection resulted in similar alterations in the neurochemistry of mice as seen in adult acquired infections. Some of these changes were observed, including tryptophan metabolism in mice exposed to the maternal immune response to T. gondii, but not congenitally infected. Both adult acquired T. gondii and congenital infection altered the brain transcriptome of mice relative to control uninfected mice with notable changes seen to transcripts of many immunologically important genes and enzymes in some of the metabolic pathways identified by LCMS. In addition, both adult acquired T. gondii infection, congenital infection and maternal exposure to different degrees were found to induce changes in a number of additional transcripts previously associated with psychoneurological diseases. These results demonstrate that maternal exposure to T. gondii infection during pregnancy induces a subset of neurochemical and transcriptomic changes found in mice with adult acquired and congenital T. gondii infection. The results therefore reinforce the potential of maternal immune activation to affect psychoneurological diseases and implicate T. gondii as a potential aetiological agent of this process.T. gondii infection acquired during life has been associated with psychoneurological disease in humans and behavioural changes in mice. However, less is known about the potential of congenitally acquired T. gondii infection, or for maternal T. gondii infection induced immune activation, to cause psychoneurological disease. The studies described herein, using LCMS (Liquid chromatography–mass spectrometry) demonstrate that adult acquired infection alters the neurochemistry and transcriptome of the brains of BALB/c mice. Notable changes to tryptophan, purine, arginine and carnitine metabolism were observed in infected mice. Congenitally infected and mice exposed to the maternal immune response to T. gondii, but not congenitally infected were found to have decreased mobility compared with control mice. Congenital T. gondii infection resulted in similar alterations in the neurochemistry of mice as seen in adult acquired infections. Some of these changes were observed, including tryptophan metabolism in mice exposed to the maternal immune response to T. gondii, but not congenitally infected. Both adult acquired T. gondii and congenital infection altered the brain transcriptome of mice relative to control uninfected mice with notable changes seen to transcripts of many immunologically important genes and enzymes in some of the metabolic pathways identified by LCMS. In addition, both adult acquired T. gondii infection, congenital infection and maternal exposure to different degrees were found to induce changes in a number of additional transcripts previously associated with psychoneurological diseases. These results demonstrate that maternal exposure to T. gondii infection during pregnancy induces a subset of neurochemical and transcriptomic changes found in mice with adult acquired and congenital T. gondii infection. The results therefore reinforce the potential of maternal immune activation to affect psychoneurological diseases and implicate T. gondii as a potential aetiological agent of this process

    The nodulin 26 aquaglyceroporin transporter : influence of transmembrane mutations, pH, calcium and phosphorylation

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    Bradyrhizobium japonicum bacteria infect the roots of soybeans resulting in the formation of a symbiotic, nitrogen-fixing nodule. Within the nodule, the bacteria are enclosed in a specialized organelle called the symbiosome. The bacteria are separated from the plant cell cytosol by the symbiosome membrane (SM). The formation of nodules is marked by the expression of several nodule-specific proteins called nodulins.In soybean, the nodulin 26 protein constitutes at least 10% of the total membrane protein and has been identified as a member of the major intrinsic protein (MIP) family. Nodulin 26 is a multifunctional aquaglyceroporin that allows the flux of both water and solutes. Besides its multifunctional transport properties, nodulin 26 has a low intrinsic water transport rate that is 50-fold lower than that of aquaporin 1, the prototypical water-transport aquaporin of the MIP family. To determine the factors that may contribute to the multifunctional transport properties of nodulin 26 and its regulation, we used the heterologous Xenopus expression system to investigate residues important for the low intrinsic transport property of nodulin 26, we tested the functional roles of AQPl-hke mutations of nodulin 26: the glutamine at position 114 of nodulin 26 was substituted by an arginine (Q114R), the cysteine at position 172 was substituted by a histidine (C172F), the valine at position 197 was substituted by a histidine (V197H), and the isoleucine at position 226 was substituted by a histidine (I226H). Findings indicate that two mutants (I226H and Q114R) are expressed and form water channels. Based on comparisons of water and glycerol permeabilities, Q114R is identical to nodulin 26 suggesting that this substitution does not confer higher water permeability or selectivity in aquaporin 1. I226H exhibits a lower permeability even though its expression appears to be normal. The other mutations (C172F and V197H) result in dysfunctional expression and/or targeting Comparison of the sequences of glycerol and water-selective aquaporins indicate that there are five discriminant residues that are invariant within each of these groups.Notable, the nodulin 26 sequence is a hybrid of the consensus sequences for aquaporins and glycerol facilitators. Weinvestigated two of these discriminant residues in nodulin26 to see if they affected its water and glycerol transport properties. The leucine at position 230 was mutated to a tryptophan (AQP-like) (L230W) and the tyrosine at position 229 was mutated to a proline (glycerol transporter-like) (Y229P). Water and glycerol permeability assays revealed that both L230W and Y229P were no longer fluxing water or glycerol above the water-injected control oocytes, again because of defects in targeting. The difficulties with improper expression, folding or trafficking to the plasma membrane made it difficult for us to draw any conclusions about the influences of these residues in the selectivity or single channel rate of nodulin 26. The low single channel rate observed for nodulin 26 suggested that nodulin 26 may undergo regulation via external signals Analysis of oocytes expressing nodulin 26 show that the relatively low water permeability of nodulin 26 is enhanced by reducing thepH. The highest water permeability was observed at pH 5.5 which was increased 3-fold compared to that observed at the standard pH of 7.6. Conversely, calcium was found to be a negative regulator of nodulin 26. A decrease in extracellular calcium increased the water permeability at least 2-fold. Conversely, an increase in external calcium exerted the opposite effect, with the relative permeability being reduced by 2-fold. Use of the calcium chelator, ethylbis(ocyethylene-nitilo)tetraacetic acid, in the recording bath further elevated the water permeability by 3-fold. The microinjection of another calcium chelator, l,2-bis(2aminophenoxy)ethane-N,N,N\u27,N\u27-tetraacetic acid, elevated the water permeability by 4-fold suggesting that the calcium sensor is internal. Previous studies indicate that nodulin 26 is the major phosphoprotein on the SM. The phosphorylation occurs at serine 262 of nodulin 26 and is catalyzed by a calcium-dependent SM-associated protein kinase of the calcium-dependent protein kinase(CDPK)family. To determine the functional effect of phosphorylation, the effects of a constitutive recombinant CDPK on the ability of nodulin 26 to transport water and glycerol using the Xenopus laevis oocytes expression system were determined. Oocytes injected with nodulin 26 alone showed an enhanced rate of oocyte swelling, whereas nodulin 26 oocytes injected with CDPK showed a 2-fold reduction in Pf. To test whether the result is specific for serine 262, a nodulin 26 mutant (serine to alanine at position 262) was generated that was unable to be phosphorylated at the 262 position. In contrast to wild-type nodulin 26, the water permeability of the alanine mutant was not affected by CDPK injection. In addition, glycerol transport properties were also made to determine if phosphorylation by CDPK affects the glycerol permeability of nodulin 26. Similar to the results obtained with the water permeability, nodulin 26 oocytes injected with CDPK showed a significant reduction in glycerol permeability compared to the nodulin 26 oocytes not injected with CDPK. Again, the alanine mutant did not show any significant reduction in glycerol permeability upon CDPK injection. Western blot analysis revealed that the decrease in nodulin 26 observed in CDPK-injected oocytes appears to be the result of a reduced level of nodulin 26 on the plasma membrane, raising the possibility that phosphorylation of nodulin 26 affected membrane trafficking. To assay the short-term effects of phosphorylation on the intrinsic transport rate of nodulin 26, the effects of phosphorylation of nodulin 26 by the endogenous Xenopus protein kinase C was determined. A synthetic peptide corresponding to the carboxyl terminus of nodulin 26, CK-15, was shown to be a protein kinase C substrate with a Kmof520 pM,suggesting that nodulin 26 is a protein kinase C substrate. To test the effect of phosphorylation on nodulin 26 activity, we used a protein kinase C agonist (phorbol-12-myristate-13-acetate, TPA) and a protein phosphatase inhibitor (okadaic acid). Both agents were shown to stimulate the water permeability of nodulin 26 by 3-fold. In contrast, mutants of nodulin 26 that do not possess a phosphorylatable residue at position 262 were not affected by either okadaic acid or TPA. Overall the data show that the water transport property of nodulin 26 was enhanced under conditions of low pH, low intracellular calcium levels, or phosphorylation. The regulation of nodulin 26 by pH, calcium, and phosphorylation may contribute to the regulation of nodulin 26 in the SM The regulation of nodulin 26 by these factors would permit the protein to shift between an activated, higher permeability state to a less active, lower permeability state to allow osmoregulation and possibly adaptation to environmental factor

    The role of the C-terminus in defining the efficiency and specificity of G-protein coupling

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    In combination with conferring resistance to ADP-ribosylation by Pertussis toxin, the substitution of a conserved cysteine residue (C351) four amino acids from the C-terminus of Gialpha1 has been shown to modulate the efficiency of coupling to the α2A adrenoceptor. Investigation of this phenomenon through systematic substitution of this cysteine residue for all other amino acids highlighted a relationship between the hydrophobicity of the substituted residue and the capacity of the a-subunit to functionally couple to the α2A adrenoceptor. From the results of this investigation, it was noted that wild type Giα1 did not display optimal coupling at this receptor. Relative to wild type Giα1, coupling was enhanced by the substitution of a more hydrophobic residue at position C351, but diminished upon the substitution of a more hydrophilic residue. In contrast to this, substitution of proline or a charged residue at this position essentially attenuated functional coupling with the α2A adrenoceptor. Similarly, pEC50 values of the mutants also showed a high degree of correlation with the hydrophobic nature of the substituted residue, with more hydrophobic residues reducing pEC50 values and more hydrophilic residues increasing pEC50 values respectively relative to wild type Giαl. This change in coupling efficiency could not be attributed to a change in the affinity for nucleotides at the a-subunit or to a change in the rate of basal guanine nucleotide exchange. These data indicate that functional coupling of the Giαl subunit to the α2A adrenoceptor is in part modulated by the physiochemical properties of residue 351 and that a relationship exists between the hydrophobicity of residue 351 and the capacity of the α-subunit to functionally couple to the α2A adrenoceptor. A series of fusion constructs composed of the α2A adrenoceptor covalently linked to selected Giαl C351 mutants were used to assess the effects of substituting residue C351 in Giαl on agonist intrinsic activity at the α2A adrenoceptor. The agonist UK 14304 was shown to elicit a spectrum of responses at the fusion constructs, closely mirroring the order of coupling efficiency previously determined in the separately expressed components. While UK14304 essentially acted as a full agonist compared to adrenaline at the fusion construct composed of wild type Giαl (C351 Giαl), it was shown to act as a partial agonist at an equivalent construct containing a glycine residue at position 351 in the Giαl moiety. In contrast to this, relative to the wild type Giαl fusion, UK14304 displayed greater relative intrinsic activity at the fusion construct containing an isoleucine residue at position 351 in the Giαl moiety. Analysis of a more extensive range of partial agonists demonstrated that the order of agonist relative intrinsic activity at the respective fusion constructs was conserved regardless of the agonist assayed. This discrepancy of intrinsic activity at the fusion proteins could not be attributed to a change in the pharmacological profile of the receptor moiety or to a modification of its affinity for agonist. These data indicate that the intrinsic activity of partial agonists, relative to adrenaline, can be modulated by the physiochemical properties of residue 351 in the Giαl moiety. The capacity of a series of chimeras containing substitutions of the last 6 C- terminal residues of Giαl for those of Gsα, Gqα and G16α were assessed for functional coupling with a range of non Gi-linked receptors. Functional coupling was demonstrated at the Gi/Gs chimera with the V2 vasopressin receptor and α2 adrenoceptor. Similarly, the Gi/Gq chimera was shown to functionally couple with the P2Y4, and TRH, receptors. No functional coupling was detected for the Gi/G16 chimera. The inability of the Gi/G16 chimera to functionally couple to any of the receptors analysed was independent from its capacity to basally exchange guanine nucleotides, which was shown to be unchanged relative to the Gi/Gs, Gi/Gq chimeras and wild type Giαl. The substitution of the 6 C-terminal residues of Giαl for those of Gsα also conferred resistance to ADP-ribosylation by both pertussis and cholera toxins. This was demonstrated by functional coupling of the Gi/Gs chimera to a FLAG(TM) -tagged version of the IP prostanoid receptor following treatment with both bacterial toxins. The GiαlGsα chimera was seen to couple more efficiently at the IP prostanoid receptor in the presence of these toxins than in non toxin treated samples, indicating that coupling efficiency of this chimera at the IP prostanoid receptor was not optimal. These findings indicate that the C-terminus of the G-protein α-subunit is an important determinant in defining G-protein/receptor coupling and that additional determinants, not present in the C-terminus of the α-subunit, are required for optimal coupling efficiency

    γ-Secretase mediated proteolytic processing of the triggering receptor expressed on myeloid cells-2 : functional implications for intracellular signaling

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    Alzheimer's disease (AD) is a progressive neurodegenerative disorder, affecting millions of people worldwide. AD is histopathologically characterized by the appearance of neurofibrillary tangles, which are intraneuronal accumulations of hyperphosphorylated tau protein, and extracellular β-amyloid plaques. β-Amyloid plaques arise from progressive accumulation of Aβ, a small hydrophobic peptide, in the brain. Aβ derives from sequential proteolytic processing of the amyloid precursor protein (APP). The final processing step in the generation of Aβ is catalyzed by the γ-secretase complex, consisting of presenilin-1 (PS1), the active subunit, nicastrin, Aph-1 (anterior pharynx defective-1) and PEN-2 (presenilin enhancer element-2). Besides APP, the γ- secretase has several other substrates and is also involved in the endocytosis of membrane bound lipoprotein receptors. In addition to neurofibrillary tangles and amyloid plaques, activation of microglia and inflammatory processes are also fundamental characteristics in the brain of AD patients. Activated microglia appear to play a dual role in AD. On one hand they produce pro-inflammatory cytokines, reactive oxygen species and nitric oxide, augmenting inflammatory processes and oxidative stress which might promote neuronal damage. On the other hand microglia can phagocytose Aβ, thereby contributing to its clearance from the brain. However, the accumulation of Aβ in AD brains indicates an insufficient clearance of Aβ in AD pathogenesis which is not yet understood. Since γ-secretase was previously linked to endocytosis, it might also be implicated in phagocytic processes by microglia and clearance of Aβ. Here it is shown by biochemical experiments in cell culture models, that the triggering receptor expressed on myeloid cells-2 (TREM2) represents a novel substrate for γ-secretase in microglia. Pharmacological inhibition of γ-secretase resulted in accumulation of a TREM2 C-terminal fragment. This fragment also accumulated upon expression of a dominant negative variant of PS1. Immunofluorescence and biotinylation experiments further indicated that the processing of TREM2 occurs at the plasma membrane. In addition, cell biological experiments demonstrated shedding of the TREM2 ectodomain. Thus, TREM2 follows the canonical proteolytic processing pathway of γ-secretase substrates which consists of an initial cleavage within the ectodomain followed by intramembranous cleavage of the resulting membrane-tethered CTF by γ-secretase. The usage of selective protease inhibitors also indicated the involvement of a metalloprotease, likely of the ADAM family, in TREM2 ectodomain shedding. TREM2 dependent signaling required the interaction with its co-receptor DAP12. Interestingly, co-immunoprecipitations revealed impaired interaction of TREM2 and DAP12 upon γ-secretase inhibition Moreover, the impaired interaction resulted in decreased phosphorylation of DAP12. Expression of different PS1 FAD mutants, led to decreased phagocytosis of Aβ. Thus, a partial loss of γ-secretase activity might decrease the capacity of microglia to clear Aβ. Taken together, these results indicate a critical function of γ-secretase in microglia and might help to understand molecular mechanisms underlying impaired Aβ clearance in the pathogenesis of AD

    Functional studies on nerve growth factor and its precursor from naja sputatrix

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    Ph.DDOCTOR OF PHILOSOPH

    Ion currents regulated by acute and chronic osmotic stimuli in rat supraoptic nucleus neurons

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    The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs. Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from –55.5 ± 6.2 to –99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration. The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing. Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration
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