京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21193号 / エネ博第367号 / 新制||エネ||72(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

No description available.

No description available.

G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function. / Die Klasse der G-protein-gekoppelten Rezeptoren (GPCRs) stellt die größte Familie membranständiger Proteine dar. GPCRs regulieren eine Vielzahl diverser physiologischer Prozesse in eukaryotischen Zellen und kontrollieren so unterschiedliche Zellfunktionen im menschlichen Organismus. Sie stellen die Zelloberflächenrezeptoren für verschiedenartige extrazelluläre Stimuli, wie zum Beispiel Photonen, niedermolekulare chemische Verbindungen, Peptide und Lipide dar. Die Wechselwirkung mit diesen sogenannten Liganden stabilisiert spezifische GPCR-Konformationen. Diese dienen wiederum als Ausgangspunkt für nachgeschaltete intrazelluläre Signalkaskaden, die beispielweise über membranverankerte G-Proteine vermittelt werden können. Während endogene GPCR-Agonisten diese Signalweiterleitung verstärken, können andere Biomoleküle wie Lipide, Ionen oder andersartige Membranproteine die Funktion, und damit die Signalweiterleitung der GPCRs modulieren. Aufgrund ihrer Einbindung in eine Vielzahl physiologischer und pathophysiologischer Prozesse, wurden GPCRs schon früh als Angriffspunkte („Targets“) zur Behandlung verschiedener Erkrankungen erforscht und genutzt. Heutzutage vermitteln etwa 30% aller zugelassenen Arzneistoffe ihre Wirkung über G-protein-gekoppelte Rezeptoren. Dennoch wird das große Potential dieser Rezeptorfamilie als Targets für medikamentöse Behandlungen noch nicht in vollem Umfang ausgeschöpft. Tatsächlich gibt es für mehr als 200 GPCRs, die nicht der olfaktorischen Wahrnehmung dienen, noch keine Arzneistoffe, da wenig über deren Pharmakologie und physiologische Bedeutung bekannt ist. Zudem wird die Entwicklung neuartiger GPCR-Liganden erheblich durch das eingeschränkte Methodenrepertoire beeinträchtigt. Alle derzeit etablierten Techniken zur Identifizierung neuer GPCR-Liganden erfassen entweder den Ligand-GPCR-Bindungsprozess, der keine Informationen über die tatsächliche Aktivität der Verbindung liefert, oder messen weit-nachgeschaltete Signale, wie Änderungen sogenannter „Second-Messenger“-Konzentrationen (meist cAMP oder Calcium) und Reporter-Gen-Expressionslevel. Aufgrund ihrer Entfernung vom eigentlichen Rezeptor-Aktivierungsprozess haben diese Methoden allerdings bedeutende Nachteile und produzieren so häufig Falsch-Positive und Falsch-Negative Ergebnisse. Seit den frühen 2000er wurden GPCR-Konformationssensoren auf Basis von Fluoreszenz-Resonanz-Energie-Transfer (FRET) zur Messung der Ligand-induzierten Rezeptordynamik genutzt. Jedoch wies keiner der bisher entwickelten FRET- oder BRET- (Biolumineszenz-Resonanz-Energie-Transfer) Sensoren ausreichende Signalstärke auf, um im Hochdurchsatz-Screening (HTS) angewendet werden zu können. Die vorliegende Studie beschreibt das erste GPCR-Sensordesign, das aufgrund seiner exzellenten Signalstärke im Hochdurchsatz-Verfahren verwendet werden kann. Wir haben 21 unterschiedliche FRET- und BRET-Sensoren des α2A-adrenergen Rezeptors (α2AAR) getestet und dabei die Kombination der kleinen und hellen Luziferase NanoLuciferase (Nluc) mit dem rot-fluoreszierenden HaloTag-Farbstoff 618 als sensitivstes RET-Paar identifiziert. Der α2AARNluc/Halo(618) Biosensor ermöglicht die Messung der Aktivität und Wirkstärke von α2AAR-Liganden im Mikrotiterplattenformat. Um die universelle Anwendbarkeit dieses Sensordesigns zu prüfen, wurden fünf weitere Nluc/Halo(618)-basierende Sensoren für GPCRs unterschiedlicher Unterfamilien entwickelt. Zudem konnten wir zeigen, dass diese GPCRNluc/Halo(618)-Fusionsproteine weiterhin ihre natürlichen Signalkaskaden in Gang setzen können und damit die biologische Funktionalität dieser Rezeptoren erhalten ist. Außerdem belegt die vorlegende Arbeit, dass diese neue Sensor-Generation zur Messung Ligand-vermittelter Rezeptordynamiken im Hochdurchsatz-Format und zur Untersuchung der GPCR-Regulation durch endogene Modulatoren genutzt werden kann. Zusammenfassend kann gesagt werden, dass wir den ersten HTS-kompatiblen Assay zur Messung der GPCR-Konformationsänderungen entwickelt haben. Diese Biosensoren erlauben die Charakterisierung neuartiger GPCR-Liganden direkt auf der Rezeptorebene und funktionieren damit unabhängig von nachgeschalteter Signalamplifikation oder Überlagerung verschiedener Signalwege, welche die Aussagekraft traditioneller GPCR-Screening-Verfahren häufig beeinträchtigen. Diese Technik kann zur Entdeckung neuartiger GPCR-Arzneistoffe genutzt werden, zu einem besseren Verständnis bisher kaum erforschter Rezeptoren beitragen und der Identifizierung und Charakterisierung potentieller GPCR-Modulatoren dienen.

In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway. In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes. The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses. / Im Herzen werden der β\(_1\)-adrenerge Rezeptor (AR) und der β\(_2\)-AR, zwei prototypische GPCR, durch die Hormone Adrenalin und Noradrenalin aktiviert. Dabei interagieren beide Rezeptoren mit dem stimulatorischen G\(_s\) Protein, bewirken eine Erhöhung des cyclischen Adenosinmonophosphates (cAMP) und beeinflussen die Kontraktionskraft und Frequenz des Herzens nach einem Stimulus. Jedoch hat die Aktivierung des β\(_1\)-ARs, nicht des β\(_2\)-ARs, auch weitere Effekte, wie z.B. Veränderungen in der Transkription von Genen. Dies wiederum führt zu Spekulationen, wie solch unterschiedliche Effekte von Rezeptoren hervorgerufen werden können, die gleiche Signalwege bedienen. In dieser Arbeit wird untersucht, ob dieses unterschiedliche Verhalten durch eine ungleiche Verteilung dieser beiden Rezeptoren in adulten Kardiomyozyten hervorgerufen werden könnte. Dazu wird die Lokalisation und die Dynamik dieser endogenen Rezeptoren in der Plasmamembran sowie im T-tubulären Netzwerk von intakten adulten Kardiomyozyten, unter Entwicklung und Verwendung hochsensitiver Fluoreszenzspektroskopiemethoden, bestimmt. Dies ermöglicht die örtliche und dynamische Eingrenzung des β\(_2\)-adrenergen Rezeptors unter endogener Expression ausschließlich auf das T-tubuläre Netzwerk. Dementgegen stellt sich heraus, dass sich der β\(_1\)-adrenerge Rezeptor ubiquitär auf der äußeren Membran und den T-Tubuli befindet und diffundiert. In β\(_2\)-AR überexprimierenden transgenen Kardiomyozyten hingegen werden diese Kompartments nicht beibehalten und es findet eine Umverteilung der Rezeptoren, auch unter Einbezug der Zelloberfläche, statt. Diese Daten können stärker darauf hindeuten, dass einige Rezeptorsubtypen sich gezielt und spezifisch bestimmte Zelloberflächen aussuchen, um somit ihre verschiedenen Signale und funktionären Effekte erzeugen zu können. Zu den Techniken, die in dieser Arbeit die Bestimmung der Lokalisation und der Dynamiken der niedrig exprimierten adrenergen Rezeptoren zulassen, gehört die Anwendung von Fluoreszenzspektroskopiemethoden in Kombination mit einem fluoreszierenden β-adrenergen Antagonisten. Weitere Techniken, die im Rahmen dieser Arbeit entwickelt wurden und in weiterführenden Studien aufschlussreiche Erkenntnisse liefern könnten, umfassen die Entwicklung eines Setups aus einer Kombination aus Weitfeld- und Konfokalmikroskopie und die Implementierung eines stabilen Autofokus mit Hilfe einer elektrisch veränderbaren Linse.

G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into a functional complex. Formation of the G protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the Gβ and Gγ subunits together. This system includes the cytosolic chaperonin containing TCP-1 (CCT) and a co-chaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and their structures determined by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. These structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ β-propeller, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. We also investigated the role of CCT and PhLP1 in folding and assembling mTOR complexes, which regulate cell growth through phosphorylation. We found that the β-propeller protein mLST8 and one of its binding partners called raptor, which is a large protein in which one domain forms a β-propeller, both bind to CCT. PhLP1 forms a ternary complex with mLST8 and CCT and may play a co-chaperone role. Depletion of PhLP1 or CCT reduces assembly of mTOR complexes in the cell. Collectively, this report reveals diversity in the contributions of CCT to the formation of protein complexes in signaling pathways and presents a molecular mechanism of Gβ folding by CCT and PhLP1.

Phosducin-like protein (PhLP), a G protein beta gamma subunit dimer binder and G protein signaling regulator, was suggested to regulate the activity of cytosolic chaperonin CCT by their high affinity interaction. In the present study, the three-dimensional structure of PhLP:CCT complex has been solved by cryoelectron microscopy. PhLP was found to bind only one of the chaperonin rings with both N- and C-terminal domains. It spans the central folding cavity of CCT and interacts with two opposite sides of the top apical region, inducing the constraining of the entry of the folding cavity. These findings support a putative role of PhLP as a co-chaperone similar to prefoldin. Docking studies with the atomic model of PhLP generated from several known structures of the homologous phosducin (Pdc) together with the immuno-EM studies have provided more details of the complex structure and predicted some regions of PhLP and the subunits of CCT involved in the interaction. Taking advantage of the fact that Pdc is highly homologous to PhLP but lack of binding to CCT, the regions of PhLP involved in the interaction with CCT were determined by testing various PhLP/Pdc chimeric proteins in the CCT binding assay. In the other part of this dissertation, the physiological role of PhLP in G protein signaling was investigated. Cellular expression of PhLP was blocked using RNA interference targeting PhLP. Together with overexpression of PhLP variants and kinetic studies of G protein beta gamma dimer formation, PhLP was determined to be a positive mediator of G protein signaling and essential for G protein beta gamma dimer expression and dimer formation. Phosphorylation of PhLP at serines 18—20 by protein kinase CK2 was required for G protein beta gamma dimer formation, while a high-affinity interaction of PhLP with CCT appeared unnecessary. Interestingly, G protein beta subunit was found to interact with CCT by co-immunoprecipitation and PhLP over-expression increased the binding of G protein beta subunit to CCT. These results suggest that PhLP and CCT act as co-chaperones in the folding and assembly of the G protein beta gamma subunit dimer by forming a ternary PhLP-Gbeta-CCT complex that is a necessary intermediate in the assembly process.

Phosducin-like protein (PhLP) has been shown to interact with the cytosolic chaperonin containing TCP-1 (CCT), and the βγ subunit dimer of heterotrimeric G proteins (Gβγ). Here we provide details obtained from cryo-electron microscopic and biochemical studies on the structure of the complex between the cytosolic chaperonin CCT and PhLP. Binding of PhLP to CCT occurs through only one of the two chaperonin rings, making multiple contacts with CCT through both its N- and C-terminal domains. In addition, we show that PhLP acts as a co-chaperonin along with CCT in mediating the assembly of the G protein βγ subunit and that assembly is dependant upon the phosphorylation of PhLP by the protein kinase CK2. Variants of PhLP lacking the CK2 phosphorylation sites, or variants with an inability to bind Gβγ block the assembly process and inhibit G protein signaling. PhLP forms a complex with CCT and nascent Gβ prior to the release of Gβγ from the ternary complex and subsequent association with the Gγ subunit to form the Gβγ dimer. In order to understand the mechanism of Gβγ dimer assembly and the role of PhLP phosphorylation in the assembly process, we provide here a method for the purification of the PhLP·CCT·Gβ ternary complex of sufficient purity for structural studies.

Chemokines are important chemical signals that guide lymphocyte movement within the immune system and promote the organization and functions of germinal centers (GCs) in the secondary lymphoid tissues. Previous studies have shown that GC T cells exhibit high expression of chemokine receptor 4, CXCR4, but that these cells are unable to migrate to the ligand for this receptor, the chemokine CXCL12. This “migratory paralysis” to CXCL12 was found to be correlated with the expression of two Regulators of G-protein Signaling, RGS13 and RGS16 in the GC T cells. The objective of my research was to determine whether RGS13 and RGS16 expression were associated with CXCL12-mediated CD4+ T cell migration. Because human GC T cells are rare and vary from one individual to another, I utilized two human neoplastic CD4+ T cell lines (i.e. Hut78 and SupT1) to facilitate and standardize my research. I also confirmed my observations using primary CD4+ T cells. Hut78 cells behaved similarly to GC T cells interms of CXCL12-mediated migration and RGS13 and RGS16 expression, while SupT1 cells appeared similar to CD4+ T cells that resided outside of GCs. The effect of RGS13 and RGS16 expression in the various CD4+ T cells was examined by altering the natural levels of these genes using RNA-mediated silencing and/or gene overexpression analysis after which, I examined the ability of the cells to migrate to CXCL12. RNA-mediated silencing of RGS16-, but not RGS13-, expression in Hut78 T cells resulted in a doubling of the migration rate in response to CXCL12. Over-expression of RGS13 or RGS16 in SupT1 and primary CD4+ T cells resulted in migration that was decreased by fifty percent. Because GC T cells demonstrated decreased migration to CXCL12 signals that may help them leave the GC, I reasoned that these cells may have an increased opportunity over other CD4+ T cells to become infected by the Human Immunodeficiency Virus (HIV) trapped on Follicular Dendritic Cells in the GCs of infected subjects. Examination of GC T cells obtained from HIV-infected subjects indicated that these cells were more frequently infected by HIV than other CD4+ T cells thereby confirming my postulate. My research indicated that RGS13 and RGS16 were associated with CXCL12-mediated CD4+ T cell migration and suggests that these molecules may play an important role in HIV pathogenesis within the GC.

G-protein coupled receptor signaling (GPCR) is essential for regulating a large variety of hormonal, sensory and neuronal processes in eukaryotic cells. Because the regulation of these physiological responses is critical, GPCR signaling pathways are carefully controlled at different levels within the cascade. Phosducin-like protein 1 (PhLP1) can bind the G protein βγ dimer and participate in GPCR signaling. Recent evidence has supported the concept that PhLP1 can serve as a co-chaperone of the eukaryotic cytosolic chaperonin complex CCT/TRiC to mediate G βγ assembly. Although a general mechanism of PhLP1-mediated G βγ assembly has been postulated, many of the details about this process are still missing. Structural analysis of key complexes that are important intermediates in the G βγ assembly process can generate snapshots that provide molecular details of the mechanism beyond current understanding. We have isolated two important intermediates in the assembly process, the Gβ1-CCT and PhLP1-Gβ1-CCT complexes assembled in vivo in insect cells, and have determined their structures by cryo-electron microscopy (cryo-EM). Structural analysis reveals that Gβ1, representing the WD40 repeat proteins which are a major class of CCT substrates, interacts specifically with the apical domain of CCTβ. Gβ1 binding experiments with several chimeric CCT subunits confirm a strong interaction of Gβ1 with CCTβ and map Gβ1 binding to α-Helix 9 and the loop between β-strands 6 and 7. These regions are part of a hydrophobic surface of the CCTβ apical domain facing the chaperonin cavity. Docking the Gβ molecule into the two 3D reconstructions (Gβ1-CCT and PhLP1-Gβ1-CCT) reveals that upon PhLP1 binding to Gβ1-CCT, the quasi-folded Gβ molecule is constricted to a more native state and shifted to an angle that can lead to the release of folded Gβ1 from CCT. Moreover, mutagenesis of the CCTβ subunit suggests that PhLP1 can interact with the tip of the apical domain of CCTβ subunit at residue S260, which is a downstream phosphorylation target site of RSK and S6K kinases from the Ras-MAPK and mTOR pathways. These results reveal a novel mechanism of PhLP1-mediated Gβ folding and its release from CCT. The next important step in testing the PhLP1-mediated Gβγ assembly hypothesis is to investigate the function of PhLP1 in vivo. We have prepared a rod-specific PhLP1 conditional knockout mouse in which the physiological consequences of the loss of PhLP1 functions have been characterized. The loss of PhLP1 has led to profound consequences on the ability of these rods to detect light as a result of a significant reduction in the expression of transducin (Gt) subunits. Expression of other G protein subunits as well as Gβ5-RGS9-1 complexes was also greatly decreased, yet all of this occurs without resulting in rapid degeneration of the photoreceptor cells. These results show for the first time the essential nature of PhLP1 for Gβγ and Gβ5-RGS dimer assembly in vivo, confirming results from cell culture and structural studies.