京都大学 / 新制・課程博士 / 博士(医学) / 甲第25489号 / 医博第5089号 / 新制||医||1073(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 林 康紀, 教授 岩田 想, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Follicle stimulating hormone (FSH) comprises an ?? subunit and a ?? subunit,whereas the FSH receptor consists of two halves with distinct functions, the N-terminalextracellular exodomain and C-terminal membrane associated endodomain. FSH initiallybinds to exodomain, and the resulting FSH/exodomain complex modulates the endodomainand generates signal. However, it has been difficult to determine which subunit of FSHcontacts the exodomain or endodomain, and in what orientation FSH interacts with them.To address these crucial issues, the receptor was Ala-scanned and the hormone subunitswere probed with photoaffinity labeling with receptor peptides corresponding to the Nterminalregion of the exodomain and exoloop 3 of the endodomain. The results show thatboth regions of the receptors are important for hormone binding and signal generation. Inaddition, the FSH ?? subunit is specifically labeled with the N-terminal peptide, whereas the?? subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH?? subunit is close to the N-terminal region and the ?? subunit is projected toward exoloop 3in the endodomain. The results raise the fundamental question whether the ?? subunit,common among the glycoprotein hormones, plays a major role in generating the hormonesignal common to all glycoprotein hormones.

Cannabis (Marijuana) has multiple effects on the human body, such as analgesia, euphoria and memory impairment. Delta-9 tetrahydrocannabinol (D9-THC), the active ingredient in cannabis, binds to cannabinoid receptors, seven-transmembrane G protein-coupled receptors (GPCRs) that mediate a variety of physiological functions. GPCRs were believed to function only in homomeric forms, however, recent findings show that different GPCRs can also form heteromeric complexes that may expand their signaling properties. In this study, we focused on Cannabinoid CB1 receptor (CB1R) heteromers with the mu-opioid receptor (MOR) and the Dopamine type 2 receptor (D2R), respectively. We utilized a variety of techniques, such as the calcium mobilization assay, a luciferase complementation assay and an electrophysiology assay to study the pharmacology of the CB1R-MOR and CB1R-D2R heteromers. Our data demonstrate that co-expression of CB1R enhances the Gi signaling through MOR and inhibits the beta-arrestin recruitment to MOR. We also show that co-application of CB1R ligands can further accentuate the MOR signaling modulation. Co-expression of a CB1R transmembrane domain 5 (TM5), but not a TM1, mini-gene abrogated the signaling change suggesting that it is likely due to heteromerization of MOR and CB1R. Utilizing this herteromeric signaling could provide a novel therapeutic approach that may yield potent analgesic effects with reduced side effects. We have also found that CB1R switched its signaling specificity from Gi to Gs upon its heteromerizaiton with D2R. In conclusion, our data show that CB1R expands its signaling repertory and modulates the partner receptor signaling upon heteromerization.

Increased signaling of various hormones through their cognate G Protein-Coupled Receptors (GPCRs), including the angiotensin, endothelin, and vasopressin systems, are implicated in human preeclampsia (PreE) and animal models of the disorder. Cascade-specific termination of GPCR signaling following receptor activation is catalyzed by the Regulator of G protein Signaling (RGS) family members. Within the RGS B/R4 family, RGS5 and RGS2 are implicated in human PreE and gestational hypertensive disorders. Mutations within the RGS2 gene, a B/R4 RGS member, are associated with human hypertensive populations and increased risk of developing PreE and its sequelae. Given the role for the placenta in the pathogenesis of PreE, we hypothesized a role for RGS2 in the placenta during PreE. My studies showed RGS2 mRNA expression is reduced in placentas from pregnancies affected by PreE. Reduced fetal-placental Rgs2 induces gestational hypertension, proteinuria, and increased plasma ALT activity in wildtype dams. Placentas with reduced Rgs2 expression exhibit reduced vascularization, increased thickness of the labyrinth and spongiotrophoblast layers, and enrichment for pathways associated with human PreE. Analysis of human PreE placenta samples reveals an increase in the cAMP/CREB signaling pathway, yet we demonstrate loss of CREB occupancy at the RGS2 promoter. HTR8 cell cultures indicate HDAC activity may be required CREB transcription of specific gene sets. In silico analysis supports this concept and further implies it may be impaired in human PreE placentas. These findings demonstrate heterozygous loss of fetal-placental Rgs2 is sufficient to induce PreE phenotypes in wildtype dams during pregnancy. Additionally, they highlight the role of the placenta in maternal pathogenesis of PreE and support the concept that paternal genetics influence the risk of developing PreE.

G protein-coupled receptors (GPCRs) are the major group of cell-surface receptors that transmit extracellular signals via classical, G protein-dependent pathways into the cell. Although GPCRs were long assumed to signal exclusively from the cell-surface, recent investigations have demonstrated a possibly completely new paradigm. In this new view, GPCR continues signaling via 3´,5´-cyclic adenosine monophosphate (cAMP) after their agonist-induced internalization of ligand/receptor complexes into an intracellular compartment, causing persistent cAMP elevation and apparently specific signaling outcomes. The thyroid stimulating hormone (TSH) receptor is one of the first GPCRs, which has been reported to show persistent signaling after ligand removal (Calebiro et al., 2009). In the meantime, signaling by internalized GPCR become a highly investigated topic and has been shown for several GPCRs, including the parathyroid hormone receptor (Ferrandon et al., 2009), D1 dopamine receptor (Kotowski et al., 2011) and beta2-adrenergic receptor (Irannejad et al., 2013). A recent study on the beta2-adrenergic receptor revealed that internalized receptor not only participates in cAMP signaling, but is also involved in gene transcription (Tsvetanova and von Zastrow, 2014). However, a biological effect of GPCR signaling at intracellular sites, which would demonstrate its physiological relevance, still remained to be shown. To investigate GPCR signaling from intracellular compartment under physiological condition, two different cellular models were utilized in the present study: intact ovarian follicles expressing luteinizing hormone (LH) receptors and primary thyroid cells expressing TSH receptors. Intact ovarian follicles were obtained from a transgenic mouse expressing, a Förster/Fluorescence Resonance Energy Transfer (FRET) sensor for cAMP to monitor cAMP/LH receptor signaling. This study provides the first accurate spatiotemporal characterization of cAMP signaling, which is derived from different cell layers of an intact ovarian follicle. Additionally, it could be shown that cAMP diffusion via gap junctions is implicated in spreading the LH-induced cAMP signals from one the outermost (mural granulosa) to the innermost (cumulus oophorus) cell layer of an ovarian follicle. Interestingly, LH receptor stimulation was associated with persistent cAMP signaling after LH removal and negligible desensitization of the cAMP signal. Interfering with receptor internalization with a dynamin inhibitor dynasore did not only prevent persistent LH-induced cAMP signaling, but also impaired the resumption of meiosis in follicle-enclosed oocytes, a key biological effect of LH. In order to investigate the downstream activation of protein kinase A (PKA) in primary thyroid cells, FRET sensors with different subcellular localization (plasma membrane, cytosol and nucleus) were transiently transfected into primary thyroid cells of wild-type mice via electroporation. Interestingly, TSH stimulation causes at least two distinct phases of PKA activation in the global primary thyroid cell, which are temporally separated by approximately 2 min. In addition, PKA activation in different subcellular compartments are characterized by dissimilar kinetics and amplitudes. Pharmacological inhibition of TSH receptor internalization largely prevented the second (i.e. late) phase of PKA activation as well as the subsequent TSH-dependent phosphorylation of CREB and TSH-dependent induction of early genes. These results suggest that PKA activation and nuclear signaling require internalization of the TSH receptor. Taken together, the data of the present study provide strong evidence that GPCR signaling at intracellular sites is distinct from the one occurring at the cell-surface and is highly physiologically relevant. / G-Protein-gekoppelte Rezeptoren (GPCR) umfassen die größte Gruppe von Rezeptoren in der Zellmembran und übermitteln extrazelluläre Signale via G-Protein-abhängige Signalwege in das Zellinnere. Obwohl lange Zeit die Wissenschaft davon ausging, das GPCR ausschließlich an der Zelloberfläche Signale weiterleiten, zeigen Studien der letzten Jahre eine vollkommen neuartige Signalweiterleitung aus dem Zellinneren. In dieser neuen Sichtweise, vermitteln GPCR nach Agonist-induzierter Internalisierung des Liganden/Rezeptor-Komplexes in das Zellinnere weiterhin zyklische Adenosin-3´,5´-monophosphat (cAMP)-Signale, was zu einer dauerhaften cAMP-Erhöhung und einem spezifischen Ergebnis der Signaltransduktion führt. Einer der ersten GPCR, für den gezeigt wurde, dass Signale aus dem Zelleninneren übertragen werden können, war der Thyreoidea-stimulierendes Hormon (TSH) Rezeptor. In der Zwischenzeit wurde die Signalübertagung von bereits internalisierten Rezeptoren für weitere GPCR gezeigt, inklusive des beta2-adrenergen Rezeptors. Vor kurzem demonstrierte eine Studie des beta2-adrenerge Rezeptors, dass die intrazellulare GPCR-Signalübertragung nicht nur an der cAMP-Weiterleitung sondern auch an der Gentranskription beteiligt ist. Bis jetzt konnte jedoch noch kein Zusammenhang zwischen der GPCR-Signaltransduktion aus dem Zellinneren und einem biologischen Effekt mit physiologischer Relevanz hergestellt werden. Um GPCR-Signaltransduktion im Zellinneren unter physiologischen Bedingungen zu untersuchen, wurden in der aktuellen Arbeit zwei unterschiedliche zelluläre Modelle verwendet: Intakte Follikel eines Ovars, welche luteinisierende Hormon (LH) Rezeptoren exprimieren und TSH-Rezeptoren-exprimierende primäre Schilddrüsenzellen. Die Follikel wurden aus einer transgenen Maus, die einen Förster/Fluoreszenz Resonanz Energie Transfer (FRET) Sensor für cAMP exprimiert, gewonnen, um cAMP/LH-Signaltransduktion zu messen. Diese Arbeit zeigt die erste exakte, zeitliche und räumliche Charakterisierung der LH- induzierten cAMP-Signaltransduktion in intakten Follikeln des Ovars. Des Weiteren konnte gezeigt werden, dass die Diffusion von cAMP via Gap Junctions ein wichtiger Bestandteil bei der Übermittlung des LH-induzierten cAMP-Signals von der äußeren (Mural granulosa) zur inneren (Cumulus oophorus) Zellebene eines Follikels darstellt. Interessanterweise ergab die LH- Rezeptor Stimulation nach Entfernung des Liganden LH ein anhaltendes cAMP-Signal sowie eine unwesentliche Desensitization des cAMP-Signals. Die Inhibition der Rezeptorendozytose mit Dynasore verhinderte nicht nur das LH-induzierte anhaltende cAMP-Signal sondern beeinflusste auch die Wiederaufnahme der Meiose durch die Follikel-eingeschlossene Oozyte, einer der wichtigsten biologischen Aufgaben von LH. Um den Einfluss der TSH-Rezeptorinternalisierung auf die PKA-Aktivität zu untersuchen, wurden primäre Schilddrüsenzellen von FVB-Mäusen, mit FRET-basierenden Protein Kinase A (PKA) Sensor exprimiert werden, via Elektroporation transfiziert. Die Ergebnisse zeigen, dass eine TSH- vermittelte Stimulation des Rezeptors mindestens zwei kinetisch und räumlich unterschiedliche PKA-Signale in Schilddrüsenzellen auslöst, die zeitlich voneinander getrennt sind. Durch die Inhibierung des TSH-Rezeptorinternalisierung konnte gezeigt werden, dass das zweite PKA-Signal sowie die darauffolgende TSH-abhängige Phosphorylierung des Trankriptionsfaktors CREB und die TSH-abhängige Regulierung von Gen Expression vermindert ist. Diese Befunde geben Aufschluss über die Notwendigkeit der Internalisierung des Rezeptors in das Zellinnere für eine effektive PKA- und Zellkern-Signaltransduktion. Zusammenfassend lässt sich sagen, dass die Ergebnisse dieser Arbeit neue, und wichtige Erkenntnisse über den Mechanismus der GPCR-Signalweiterleitung im Zellinneren und erstmals einen Einblick über die biologische Relevanz der Rezeptorinternalisierung liefern.

Myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis was the only previously identified myokinin receptor. A cDNA encoding a neuropeptide receptor was cloned from the southern cattle tick, Boophilus microplus. The deduced amino acid sequence was 40 % identical to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative RT-PCR. When expressed in mammalian CHO-K1 cells, myokinins at nanomolar concentrations induced increases in intracellular calcium as measured by fluorescent cytometry. The rank order of potency for peptides tested was FFFSWS-NH2≥FFFSWG-NH2≥FFSWG-NH2>FYSWG-NH2>muscakinin>lymnokinin>>APTGFFGVR-NH2. The receptor coupled to a pertussis toxin insensitive G protein. Absence of extracellular calcium did not inhibit the calcium response, indicating the release of Ca2+ from intracellular stores. Receptor transcript was detected by RT-PCR in the dissected synganglia, ovaries, salivary glands, guts and Malpighian tubules of partially engorged adult female ticks. It is concluded that the B. microplus receptor is the first myokinin receptor cloned from an arthropod, and the first neuropeptide receptor known from the Acari. The presence of this receptor transcript in multiple tissues and all life stages suggests a multifunctional role in ticks.

M2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemmal membranes. Whereas the latter has been shown to exhibit non-competitive effects in the binding of N-methylscopolamine (NMS) and quinuclidinylbenzilate (QNB), which can be explained in terms of cooperativity within a receptor that is at least tetravalent, binding to the former is essentially competitive. Levels of cholesterol in Sf9 membranes were only 5% of those in sarcolemmal membranes and were increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M2 receptors extracted from CHL-treated Sf9 membranes resembled those from heart; that is, cholesterol induced a pronounced heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were marked discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites, apparently within an oligomer. Cholesterol also was found to increase the affinity of the receptor for NMS and QNB, and the effect was examined for its possible relationship to the known interconversion of cardiac muscarinic receptors between an agonist-specific (R*) and an antagonist-specific (R) state. Cholesterol and N-ethylmaleimide (NEM) were compared for their effect on the affinity of NMS, QNB and four muscarinic agonists, and the data were assessed in terms of an explicit mechanistic model for a receptor that interconverts spontaneously between two states. The data can be described equally well by an effect of cholesterol on either the distribution of receptors between R and R* or the affinity of all ligands for both states, with an accompanying effect of NEM on either the affinity or the distribution between states, respectively. Since NEM is known from other data to favor R* over R, cholesterol appears to increase affinity per se. Cholesterol therefore is a determinant of affinity and cooperativity in the binding of orthosteric ligands to the M2 receptor. Both effects are observed in solution and therefore appear to arise from a direct interaction between the lipid and the receptor.

M2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemmal membranes. Whereas the latter has been shown to exhibit non-competitive effects in the binding of N-methylscopolamine (NMS) and quinuclidinylbenzilate (QNB), which can be explained in terms of cooperativity within a receptor that is at least tetravalent, binding to the former is essentially competitive. Levels of cholesterol in Sf9 membranes were only 5% of those in sarcolemmal membranes and were increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M2 receptors extracted from CHL-treated Sf9 membranes resembled those from heart; that is, cholesterol induced a pronounced heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were marked discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites, apparently within an oligomer. Cholesterol also was found to increase the affinity of the receptor for NMS and QNB, and the effect was examined for its possible relationship to the known interconversion of cardiac muscarinic receptors between an agonist-specific (R*) and an antagonist-specific (R) state. Cholesterol and N-ethylmaleimide (NEM) were compared for their effect on the affinity of NMS, QNB and four muscarinic agonists, and the data were assessed in terms of an explicit mechanistic model for a receptor that interconverts spontaneously between two states. The data can be described equally well by an effect of cholesterol on either the distribution of receptors between R and R* or the affinity of all ligands for both states, with an accompanying effect of NEM on either the affinity or the distribution between states, respectively. Since NEM is known from other data to favor R* over R, cholesterol appears to increase affinity per se. Cholesterol therefore is a determinant of affinity and cooperativity in the binding of orthosteric ligands to the M2 receptor. Both effects are observed in solution and therefore appear to arise from a direct interaction between the lipid and the receptor.

La rodopsina es el fotopigmento de los bastones, las células fotorreceptoras de la retina de los vertebrados. Esta proteína es un receptor de siete hélices transmembranales que pertenece a la familia de los receptores acoplados a proteína G (GPCR). Se han estudiado mutantes puntuales de la rodopsina relacionados con la Retinosis pigmentaria (RP), una enfermedad degenerativa progresiva de la retina, utilizando técnicas de mutagénesis dirigida y técnicas espectroscópicas. Esto ha permitido aportar nuevos conocimientos sobre el estudio de la estructura-función en la rodopsina. Hemos realizado el análisis de varias mutaciones asociadas a RP, localizadas en los diferentes dominios de la proteína: transmembranal, intradiscal y citoplasmático. Por otro lado se ha llevado a cabo un estudio detallado de diferentes mutaciones en la posición 125, asociada a RP, para aportar información precisa sobre la implicación de este residuo en la estructura y el mecanismo de activación del receptor. Mediante espectroscopia de absorción en el UV-Vis se ha determinado la capacidad de regeneración de las proteínas con el 11-cis-retinal. La capacidad de los mutantes de activar la proteína G ha sido determinada mediante espectroscopia de fluorescencia. Las mutaciones de RP G106W, G114D, P171Q y H211R provocan un mal plegamiento de las proteínas ya que impiden la regeneración con el cromóforo. Los mutantes de rodopsina M44T, R135L, V137M y L328P pueden unir 11-cis-retinal pero presentan una activación de la proteína G alterada. El conjunto de resultados obtenidos de la caracterización de los mutantes en la posición 125 situada próxima al anillo de la b-ionona indican que es clave para mantener la estructura del bolsillo de unión del cromóforo. Las sustituciones en esta posición pueden alterar la conformación óptima del bolsillo del retinal y consecuentemente la estructura y señalización del receptor. Estos resultados obtenidos a nivel molecular se discuten en relación con los procesos degenerativos de la retina observados en la RP. / Rhodopsin is the visual pigment of rod phototoreceptor cells in the vertebrate retina which mediate dim light vision. This protein is a seven transmembrane helical receptor belonging to the G-protein-coupled receptor (GPCR) superfamily. We have studied point mutations in rhodopsin associated with the retinal degenerative disease retinitis pigmentosa (RP) by using a combination of molecular biology and biophysical techniques, namely site-directed mutagenesis and spectroscopic techniques. This approach has allowed new insights into structure-function relationships in rhodopsin to be obtained. We have expressed and characterised mutations associated with RP in the different domains of the protein: transmembrane, intradiscal and cytoplasmic. In addition, a set of mutations at position 125 (site of the Leu-125-Arg RP mutation) have also been studied to further investigate the role of Leu 125 in the correct folding and function of rhodopsin. The ability of the proteins to bind 11-cis-retinal was determined by means of UV-Vis spectroscopy, and the functionality of the mutant proteins was determined from its ability to activate the G protein transducin, as measured by fluorescence spectroscopy. The RP-related mutations, G106W, G114D, P171Q and H211R cause misfolding of the mutant proteins, this resulting in impaired 11-cis-retinal binding and chromophore regeneration. Rhodopsin mutants M44T, R135L, V137M and L328P regenerated in the presence of 11-cis-retinal but its G-protein activation ability was altered. The results from the characterisation of mutants at position 125, located close to the b-ionone ring of the retinal chromophore, indicated that this position is important in maintaining the correct structure of the retinal binding-pocket. Substitutions at this position may alter the structure of the binding-pocket and, consequently, the correct structure and signalling mechanism of the receptor. The possible correlation between the molecular results obtained from the mutant proteins studied, and the clinical progression observed in patients affected by RP, is discussed.

Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.