archives@tulane.edu / Glucocorticoid-induced negative feedback of the hypothalamic-pituitary-adrenal axis is rapidly achieved by two mechanisms: desensitization of the corticotropin releasing hormone-producing neurons to excitatory noradrenergic inputs by internalization of the α-1 receptor and suppression of excitatory synaptic input via production of an endocannabinoid retrograde messenger. Several previously undetermined signaling factors in the glucocorticoid-induced endocannabinoid release were identified here. The glucocorticoid-induced desensitization to adrenergic receptor signaling was revealed to involve α-1 receptor intracellular trafficking to the late endosome. The physiological significance and therapeutic targets of attenuation of the stress response to noradrenergic inputs was also investigated using electrophysiology and pharmacogenetics. These experiments indicate that desensitization to norepinephrine selectively attenuates the stress response to physiological over psychological stressors. These rapid interactions between glucocorticoids and adrenoreceptor trafficking and endocannabinoid synthesis represent novel glucocorticoid signaling mechanisms through G-protein coupled receptors. / 1 / Grant L. Weiss

No description available.

The study of arrestins as regulators of seven transmembrane receptor (7TMR) signaling has revealed multiple levels of complexity, initiating desensitization of G protein activity and coordination of receptor internalization via clathrin‐coated pits. Recently, β‐arrestins have also been shown to act as adaptor proteins, mediating G protein‐independent signaling as well as scaffolding of enzymes that degrade second messenger molecules. This latter function was demonstrated by β‐arrestins recruiting PDE4 phosphodiesterase to Gs‐coupled β2‐adrenergic receptors, enhancing metabolism of the second messenger cAMP. As β‐arrestins universally interact with members of the 7TMR superfamily, we sought to determine if this phenomenon of concerted desensitization might be applicable to additional receptor subtypes. We screened for β‐arrestin‐binding proteins among modulators of diacylglycerol and IP3 (second messengers downstream of Gq‐coupled 7TMRs). We observed β‐ arrestins constitutively interacted with members of the diacylglycerol kinase (DGK) family, which phosphorylate diacylglycerol to create phosphatidic acid. Furthermore, examining lipid extracts of 32P labeled cells separated by TLC, we observed that overexpression of β‐arrestin enhanced phosphatidic acid (PA) production after M1 muscarinic receptor stimulation. Conversely, depletion of β‐arrestins by RNA interference showed significantly decreased agonist‐stimulated PA accumulation. Additionally, overexpression of a β‐arrestin2 mutant that binds DGKs but not receptors served as a dominant negative for agonist‐dependent DGK activity. These results demonstrate a requirement for β‐arrestins in DGK translocation to the membrane, and specifically to activated 7TMRs, where concentrations of second messengers are at their highest. Phosphatidic acid is an effector for several enzymes, including the phosphatidylinositol 5‐kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus, we hypothesized β‐arrestin‐targeted DGKs may regulate PIP5K activity. PIP5K Iα associated with β‐arrestin2 in an agonist‐dependent manner in HEK293 cells, and a β‐ arrestin2 mutant defective in receptor endocytosis (a PIP2‐dependent function) was impaired. Furthermore, knockdown of β‐arrestin2 by RNAi significantly decreased the amount of PIP5K Iα detected in receptor immunoprecipitates. In TLC assays, overexpressing both β‐arrestin2 and PIP5K Iα enhanced agonist‐stimulated PIP2 labeling, while either protein alone had no effect. These data support the concept of β‐ arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which then stimulates production of PIP2, promoting receptor internalization. / Dissertation

<p>Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby, in turn, instruct its functional capabilities. </p><p>We determined conformational changes in beta-arrestin1 in vitro using limited tryptic proteolysis and MALDI-TOF MS analysis in the presence of a phosphopeptides derived from the C-terminus of the V2 vasopressin receptor (V2Rpp or V2R4p) or the corresponding unphosphorylated peptide (V2Rnp). Upon V2Rpp binding, we show that the previously shielded R393 becomes accessible, which indicates release of the C-terminus. Moreover, we have shown that R285 becomes more accessible and this residue is located in a region of &beta;-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1. We also show a functional consequence of the release of beta-arrestin1's C-terminus by enhanced clathrin binding. In addition, we have shown marked protection of beta-arrestin1's N-domain in the presence of V2Rpp; consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. Using a differentially phsophorylated V2R peptide (V2R4p), we show that beta-arrestin1 is able to adopt distinct conformations in response to different phosphorylation patterns. Futhermore, a striking difference is observed in the conformation of V2Rpp-bound beta-arrestin1 when compared to beta-arrestin2, namely the flexibility of the inter-domain hinge region. These data represent the first direct evidence that the beta-arrestin1 conformation is differentially instructed by phosphorylation patterns and that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.</p><p>Phosphorylation of 7TMRs by GRKs plays essential roles in regulation of receptor function by promoting interactions of the receptors with beta-arrestins. We hypothesized that different GRKs phosphorylate distinct sets of sites thereby establishing a "bar code." In order to test this hypothesis, we monitored the phosphorylation events of the beta2AR upon stimulation with a classical full agonist, isoproterenol, or a beta-arrestin "biased" agonist, carvedilol, in the presence of a full complement of GRKs or when individual GRKs (2 or 6) were depleted by siRNA. We demonstrate that at least thirteen sites on the beta2AR show changes in phosphorylation in response to the agonist isoproterenol. Of these, phosphorylation increased 10 to more than 300 fold in 12 (S261, S262, S345, S346, S355, S356, T360, S364, S396, S401, S407 AND S411) and decreased 50% in one (S246). Depletion of GRK2 or 6 by siRNA indicates that S355, 356 are GRK6 sites whereas the remainder are GRK2 sites. Phosphorylation of GRK2 sites inhibits that of GRK6 sites. Carvedilol, a beta-arrestin biased agonist, promotes phosphorylation of only the GRK6 sites S355, 356. In HEK293 cells, GRK2 phosphorylation is found to be the major positive regulator of receptor internalization; to contribute to receptor desensitization; and to inhibit beta-arrestin mediated ERK activation. Phosphorylation of the two GRK6 sites contributes to receptor desensitization and internalization and is required for beta-arrestin mediated ERK activation. These data indicate that different ligands promote distinct patterns of receptor phosphorylation which dictate different patterns of beta-arrestin mediated function.</p> / Dissertation

Die Aufgabe des Proteins Arrestin ist die Beendigung der Signalweitergabe über den GPCR Signalweg. In Stäbchenzellen bindet Arrestin an Licht-aktiviertes phosphoriliertes Rhodopsin um die Signalweitergabe zu unterdrücken. Die Bindung von Arrestin an Rhodopsin erfolgt in zwei Schritten. Zunächst wechselwirkt Arrestin mit dem phosphorilierten C-Terminus von Rhodopsin und bildet einen prä-Komplex, dies induziert Konformationsänderungen im Arrestin wodurch die Bildung eines High-affinity Komplex unter Kopplung an den helikalen Kern des aktivierten Rezeptors erfolgen kann. Biochemische Untersuchungen und Kristallstrukturen haben einen Einblick in die Konformation des Komplexes aus Arrestin und Rhodopsin ermöglicht. In dieser Arbeit werden site-directed Fluorezenz Experimente angewandt um die strukturellen Änderungen zu untersuchen, die bei der Bindung von Arrestin an Rhodopsin ablaufen und der nterschiedlichen Bindungsmodi innerhalb der Wechselwirkung zwischen Arrestin und Rhodopsin. Insbesondere wird hier eine, bisher nicht beschriebene, Assoziation von Arrestin an die Membran untersucht. Des Weiteren wurden Erkenntnisse über die Struktur des prä-Komplexes gewonnen. Die Konformation vom Arrestin im prä-Komplex scheint die Konformation im Basalzustand nachzubilden unter Beteiligung zweier Kontaktstellen: Interaktion mit dem phosphorilierten C-Terminus des Rezeptors und Assoziation mit der Membran. Beim Übergang in den High-affinity Komplex durchläuft Arrestin eine Konformationsänderung in eine aktivere Konformation: der C-Terminus wird verdrängt, es erfolgt eine Neuausrichtung der zentralen flexiblen Schleifen und die Orientierung des Membranankers ändert sich. Die Aufgabe des prä-Komplexes ist somit Arrestin und den Rezeptor zusammen zu bringen sowie die korrekte Orientierung sicherzustellen um einen schnellen Übergang in den High-affinity Komplex zu ermöglichen. / The protein arrestin is responsible for termination of GPCR signalling. In the rod cell, arrestin binds light-activated phosphorylated rhodopsin in order to block further signal transduction. The binding of arrestin to rhodopsin is a two-step process. Arrestin first interacts with the phosphorylated receptor C-terminus in a pre-complex, which induces conformational changes in arrestin that allow coupling to the helical core of the active receptor in a high-affinity complex. Biochemical studies and crystal structures have provided insights into the conformation of the arrestin-rhodopsin complex. This dissertation describes site-directed fluorescence experiments, which were carried out to further investigate the conformational changes occurring upon arrestin binding to rhodopsin and the nature of different binding modes of the arrestin-rhodopsin interaction. In particular this involved characterization of a previously unidentified association of arrestin with the membrane, as well as further elucidation of the structure of the pre-complex. The conformation of arrestin in the pre-complex is indicated to resemble that of the basal state of arrestin, and involves two sites of contact: interaction with the phosphorylated receptor C-terminus, and association with the membrane. Upon transition to the high-affinity complex, arrestin undergoes a conformational change to a more active conformation: the auto-inhibitory C-tail is displaced, there is movement within the central flexible loops, and the orientation of the membrane anchor changes. The pre-complex therefore most likely functions to bring arrestin and the receptor into close contact, and in the correct orientation, to allow for fast transition to the high-affinity complex.

<p>Sonic hedgehog (Shh) is a critical secreted signaling molecule that regulates many aspects of organogenesis. In the absence of Shh, many organs, including the foregut, larynx, palate, cerebellum and heart do not form properly. However, the cellular details of the roles of Shh, including the relevant domains of Shh expression and reception, have not been elucidated for many of these processes. </p><p>The single embryonic foregut tube must divide into the trachea and esophagus, which does not occur in the Shh-null mutant. In Chapter 5, I use Cre-Lox technology to determine that the ventral foregut endoderm is the relevant source of Shh for this process and the mesoderm must directly receive that Shh signal. Surprisingly, this signaling event appears to occur two days before the foregut begins to divide, indicating an early essential role for Shh in foregut division. </p><p>Shh is also expressed at later stages in the maturing trachea and esophagus. In Chapter 6, I demonstrate that these domains serve to establish differentiated mesoderm. In the trachea, Shh from the endoderm signals directly to the mesoderm to form the tracheal cartilage rings. In the esophagus, the roles of Shh are more complex. Shh regulates the size of the esophagus and controls patterning of the concentric rings of esophageal mesoderm, however this process seems to be indirect, requiring autocrine Shh signaling within the esophageal endoderm. </p><p>The laryngeal apparatus is entirely absent in the Shh-null mouse. I n Chapter 3, I dissect the domains of Shh expression and reception required for laryngeal development and demonstrate that loss of endodermal Shh expression causes laryngotracheoesophageal clefts and malformed laryngeal cartilages. As much of laryngeal morphogenesis poorly understood, I also utilize dual mesodermal and neural crest fate maps to determine the embryonic origins of various laryngeal tissues. Finally, as Shh signaling often occurs in concert with Bone Morphogenic Protein (BMP) signaling, I investigate the roles of BMP signaling in laryngeal development. </p><p>Much of Shh signaling occurs at the primary cilium, to which Smoothened, a critical pathway member, must translocate upon Shh signal transduction. This process requires a Smo-Kif3a-&#946;arretin complex in mammalian cell culture. However, the roles of &#946;arrestins in mouse development, and their relationship to Shh signaling have not been investigated in vivo. To do so, in Chapter 4, I analyze the phenotypes of the &#946;arr1/&#946;arr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling. </p><p>Altogether, my work dissects the cellular details of Shh signaling during multiple organ systems in the mouse embryo. I further analyze the consequences of absent or misregulated Shh signaling across multiple developmental contexts and determine that Shh plays critical and diverse roles in organogenesis.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 107-117).

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23366号 / 医博第4735号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 萩原 正敏, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die phosphorylierungsabhängige Bindung von Arrestin an G-Protein-gekoppelte Rezeptoren (GPCRs) ist ein weit verbreiteter Mechanismus, um aktive Rezeptoren zu inhibieren. Für die Bindung von Arrestin an lichtaktiviertes, phosphoryliertes Rhodopsin (pRho), einem GPCR, der sich in der Diskmembran der Stäbchenzellen der Netzhaut befindet, ist in Lipidmembranen die Phosphorylierung von 2 der insgesamt 7 Phosphorylierungsstellen im Rezeptor-C-Terminus nötig, wohingegen 3 Phosphate das Arrestin quantitativ aktivieren. Welchen Einfluss höhere Phosphorylierungsniveaus auf die Rezeptor-Arrestin-Interaktion haben, ist bisher ungeklärt. In dieser Arbeit wurde daher eine Methode zur quantitativen Bestimmung der Rhodopsin-Phosphorylierung etabliert, die unterschiedlich pRho-Spezies präparativ getrennt und mit den isolierten pRho-Spezies verschiedene Parameter der Rezeptor-Arrestin-Interaktion, wie z.B. die Bindungsstöchiometrie der resultierenden pRho-Arrestin-Komplexe, untersucht. Für die Charakterisierung der Arrestinbindung wurden Titrationen mit 3 verschiedenen fluoreszenzmarkierten Arrestinmutanten und pRho in gemischten Phospholidipd/Detergens-Mizellen durchgeführt. Die in dieser Arbeit gewonnenen Daten zeigen, dass der Phosphorylierungsgrad von Rhodopsin neben der Affinität von Arrestin auch die Bindungsstöchiometrie beeinflusst. So haben die Komplexe bei geringem Phosphorylierungsgrad eine 2 : 1 (pRho : Arrestin) Stöchiometrie, während bei einem sehr hohen Phosphorylierungsgrad bevorzugt 1 : 1 pRho-Arrestin-Komplexe gebildet werden. Zudem weisen ein unterschiedliches Elutionsverhalten bei der säulenchromatographischen Trennung und Abweichungen in der pRho-Arrestin-Stöchiometrie verschiedener pRho-Präparationen mit ähnlicher Gesamtanzahl an Phosphaten auf einen zusätzlichen Einfluss unterschiedlicher Phosphorylierungsmuster hin. Insgesamt deuten die Daten auf eine komplexe Beziehung zwischen dem Phosphorylierungsgrad der Rezeptoren und dem Arrestin-Bindungsmodus hin. / The phosphorylation-dependent binding of arrestin to G protein-coupled receptors (GPCRs) is a widely used mechanism to inhibit active receptors. In lipid membranes, the binding of arrestin to light-activated, phosphorylated rhodopsin (pRho), a GPCR located in the disc membranes of retinal rod cells, requires the phosphorylation of 2 of the 7 phosphorylation sites in the receptor C-terminus, whereas 3 phosphates are required to completely activate arrestin. The influence of higher phosphorylation levels on the receptor/arrestin interaction is still unclear. In this thesis, a method for the quantitative determination of rhodopsin phosphorylation was established, rhodopsin species with varying degrees of phosphorylation were separated preparatively and different parameters of the receptor/arrestin interaction, such as the binding stoichiometry of the resulting pRho/arrestin complexes, were investigated for each isolated pRho species. For the characterization of arrestin binding, titrations with 3 different fluorescently labeled arrestin mutants and pRho in mixed phospholipid/detergent micelles were performed. The data obtained in this work show that the phosphorylation level of rhodopsin influences the binding stoichiometry in addition to the affinity of arrestin binding to rhodopsin. Thus, complexes with a low level of phosphorylation have a 2 : 1 (pRho : arrestin) stoichiometry, whereas 1 : 1 pRho/arrestin complexes are preferably formed at a very high degree of phosphorylation. In addition, a different elution behaviour during chromatographic separation and variances in the pRho/arrestin stoichiometry of different pRho preparations with similar overall number of phosphates indicate an additional influence of distinct phosphorylation patterns. Overall, the data indicate a complex relationship between receptor phosphorylation level and arrestin binding mode.

Beta-arrestins have been suggested to regulate μ-, δ-, and κ-opioid receptor-mediated responses. In the present study, we examined the effects of pretreatment with β-arrestin-2 antibody on tail-flick inhibition induced by opioid receptor agonists in the mouse spinal cord. Intrathecal (i.t.) pretreatment with β-arrestin-2 antibody potentiated the antinociception induced by i.t.-administered μ-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) and endomorphin-1, but not endomorphin-2, the δ-opioid receptor agonist [D-Ala2]deltorphin II or the κ-opioid receptor agonist U50,488H. The present result suggests that β-arrestin-2 may tonically down-regulate a selected population of μ-opioid receptors activated by endomorphin-1 or DAMGO in the mouse spinal cord.