Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24525号 / 医博第4967号 / 新制||医||1065(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 渡邊 直樹, 教授 中川 一路, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

The D3 dopamine receptor (D3R) belongs to D2-class of dopamine receptors (DARs) and is involved in emotion, movement, and reward. D3R dysfunction has been reported in some neuropsychiatric disorders such as addiction, cognitive deficits, depression, schizophrenia, and Parkinson’s disease. Genetic studies have shown two polymorphic variants of the D3R gene resulting from substitution of serine to glycine at position nine of the amino terminus. Isoform 5 of adenylyl cyclase (AC5) is one of the nine transmembrane bound ACs in the brain and myocardium. Previous studies in rats have shown that AC5 is expressed in the striatum, nucleus accumbens and olfactory tubercle and at lower levels in islands of Calleja, where the D3R is also expressed. Previous studies showed that although D2R and D4R inhibit ACs activity in different cell types, inhibition of ACs by D3R is weak and often undetectable. It has been shown that D3R selectively inhibits AC5 activity in human embryonic kidney 293 (HEK293) cells co-transfected with D3R and AC5. Co-expression of D3R and AC5 in brain regions which are major coordinators of normal and pathological movement, and the selective inhibition of AC5 activity by D3R raise the possibility of a functional link between AC5 and D3R in the modulation of signal transduction and trafficking. I hypothesized that AC5 plays a unique role in modulation of D3R trafficking and signaling pathways through interaction between D3R and AC5. Herein, I demonstrated an interaction between D3R and AC5 in vivo and in vitro using reciprocal co-immunoprecipitation/immunoblotting (co-IP/IB) assays. Interestingly, DA may facilitate the formation of protein complex between D3R and AC5 in vitro. Radio ligand binding assays revealed that heterodimerization of D3R polymorphic variants with AC5 does not change ligand binding affinity and expression of the D3R. Furthermore, taking advantages of GloSensor assays, selective inhibition of AC5 activity by D3Ser9 and D3Gly9 has been shown following activation by DA and quinpirole. Using ELISA studies showed that AC5 promotes cell surface expression and total expression of D3Ser9 and D3Gly9. Moreover, ELISA results suggested that AC5 facilitates DA-induced D3Ser9 endocytosis in dynamin and β-arrestin 2 dependent process, while having no effect on D3Gly9 polymorphic variant. The results also revealed that AC5 attenuates heterologous (PKC-induced) internalization of D3Ser9, while it does not have any effect on D3Gly9 heterologous internalization. My results also displayed a complex formation between D3R, AC5 and, β-arrestin 2 under basal and DA stimulation conditions, which emphasize the role of β-arrestin 2 in D3R signal transduction. Overall, a new regulatory mechanism for D3R has been suggested. My results suggested that complex formation between both D3R polymorphic variants with AC5 can regulate signaling and trafficking properties of D3R without changing the binding affinity of the receptor. These data will be meaningful for understanding of diseases and developing treatment strategies.

Récemment plusieurs récepteurs couplés aux protéines G (RCPGs) ont été caractérisés au niveau des membranes intracellulaires, dont la membrane nucléaire. Notre objectif était de déterminer si les sous-types de récepteurs β-adrénergiques (βAR) et leurs machineries de signalisation étaient fonctionnels et localisés à la membrane nucléaire des cardiomyocytes. Nous avons démontré la présence des β1AR et β3AR, mais pas du β2AR à la membrane nucléaire de myocytes ventriculaires adultes par immunobuvardage, par microscopie confocale, et par des essais fonctionnels. De plus, certains partenaires de signalisation comme les protéines GαS, Gαi, l’adénylate cyclase II, et V/VI y étaient également localisés. Les sous-types de βAR nucléaires étaient fonctionnels puisqu'ils pouvaient lier leurs ligands et activer leurs effecteurs. En utilisant des noyaux isolés, nous avons observé que l'agoniste non-sélectif isoprotérénol (ISO), et que le BRL37344, un ligand sélectif du β3AR, stimulaient l'initiation de la synthèse de l’ARN, contrairement à l'agoniste sélectif du β1AR, le xamotérol. Cette synthèse était abolie par la toxine pertussique (PTX). Cependant, la stimulation des récepteurs nucléaires de type B de l’endothéline (ETB) causaient une réduction de l'initiation de la synthèse d’ARN. Les voies de signalisations impliquées dans la régulation de la synthèse d’ARN par les RCPGs ont ensuite été étudiées en utilisant des noyaux isolés stimulés par des agonistes en présence ou absence de différents inhibiteurs des voies MAP Kinases (proteines kinases activées par mitogènes) et de la voie PI3K/PKB. Les protéines impliquées dans les voies de signalisation de p38, JNK, ERK MAP Kinase et PKB étaient présents dans les noyaux isolés. L'inhibition de PKB par la triciribine, inhibait la synthèse d’ARN. Nous avons ensuite pu mettre en évidence par qPCR que la stimulation par l’ISO entrainait une augmentation du niveau d'ARNr 18S ainsi qu’une diminution de l'expression d’ARNm de NFκB. En contraste, l’ET-1 n’avait aucun effet sur le niveau d’expression de l’ARNr 18S. Nous avons ensuite montré que la stimulation par l’ISO réduisait l’expression de plusieurs gènes impliqués dans l'activation de NFκB, tandis que l’inhibition de ERK1/2 et PKB renversait cet effet. Un microarray global nous a ensuite permis de démontrer que les βARs et les ETRs nucléaires régulaient un grand nombre de gènes distincts. Finalement, les βARs et ETRs nucléaires augmentaient aussi une production de NO de noyaux isolés, ce qui pouvait être inhibée par le LNAME. Ces résultats ont été confirmés dans des cardiomyocytes intacts en utilisant des analogues cagés et perméables d’ISO et de l'ET-1: l'augmentation de NO nucléaire détectée par DAF2-DA, causée par l'ET-1 et l'ISO, pouvait être prévenue par le LNAME. Finalement, l’augmentation de l’initiation de la transcription induite par l'ISO était aussi bloquée par le L-NAME ou par un inbitheur de PKG, le KT5823, suggérant que la voie NO-GC-PKG est impliquée dans la régulation de la transcription par les βAR. En conclusion, les βARs et les ETRs nucléaires utilisent des voies de signalisation différentes et exercent ainsi des effets distincts sur l’expression des gènes cardiaques. Ils représentent donc une avenue intéressante pour le développement de drogues pharmacologiques. / Recently several G protein-coupled receptors (GPCRs) have been shown to localize to intracellular membranes, in particular the nuclear membrane. As such, we sought to determine if the β-adrenergic receptor (βAR) subtypes and their associated signalling machinery are functionally localized to nuclear membranes. We demonstrated the presence of β1AR and the β3AR, but not the β2AR, in adult ventricular myocyte nuclei by western blotting, confocal microscopy and functional assays. Downstream signalling partners such as GαS, Gαi and adenylyl cyclase II and V/VI were also present. Nuclear-localized βARs were functional with respect to ligand binding and effector activation. In isolated nuclei, the non-selective βAR agonist isoproterenol (ISO) and the β3AR-selective ligand BRL37344, but not the β1AR-selective xamoterol, stimulated transcription initiation in a pertussis toxin (PTX)-sensitive manner. In contrast, stimulation of type B endothelin receptors (ETB), another GPCR family shown to be present on the nuclear membrane, decreased de novo RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of the PI3K/PKB and mitogen-activated protein kinase (MAPK) pathways. Components of p38, JNK, and ERK1/2 MAPK cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine converted the activation of the βAR from stimulatory to inhibitory with regards to transcription initiation. Analysis by qPCR indicated isoproterenol treatment increased 18S rRNA but decreased NFκB mRNA. In contrast, ET-1 had no effect on 18S rRNA expression. Further investigation using pathway-specific PCR arrays revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB and that ERK1/2 and PKB inhibitors attenuated this effect. Subsequent genome-wide microarray analysis has revealed that nuclear βAR and ETB regulated a host of genes in an overlapping but distinct manner. Moreover, both ET-1 and ISO produced an L-NAME-sensitive increase in NO production in isolated cardiac nuclei. These observations were confirmed in intact cardiomyocytes using novel caged analogues of ISO and ET-1 and the cell-permable NO-sensitive fluorescent dye, DAF-2 DA. Briefly, both ET-1 and isoproterenol increased NO production, and this increase was prevented upon preincubation with L-NAME. Moreover, the ability of isoproterenol to increase transcription initiation in isolated nuclei was blocked by L-NAME or the PKG inhibitor KT5823, indicating the NO-GC-PKG pathway is involved in the regulation of gene expression by nuclear βARs. Hence, we have shown that βARs and ETRs in the nuclear membrane activate distinct signalling pathways, resulting in different effects on gene transcription and thus represent potentially important targets for drug development.

In der vorliegenden Arbeit wurden die molekularen Grundlagen für die Agonistspezifität des G-Protein-gekoppelten Rezeptors GPR34 untersucht. Mittels verschiedener funktioneller Versuche konnte an ausgewählten Orthologen des Rezeptors gezeigt werden, dass, im Gegensatz zu publizierten Daten, Lysophosphatidylserin (Lyso-PS) nicht der natürliche Agonist des GPR34 ist. Lediglich an einigen cyprinoiden Subtypen des GPR34 hat Lyso-PS surrogat-agonistische Effekte. Anhand eines detaillierten evolutionären Vergleichs von Orthologen konnten Bereiche des Rezeptors ermittelt werden, welche an der Ligandenbindung, und damit an der Agonistspezifität des GPR34 beteiligt sind. Durch Übertragung dieser Bereiche vom Karpfen-GPR34-Subtyp 2a auf den humanen GPR34 konnte dieser zu einem Lyso-PS-sensitiven Rezeptor modelliert werden. Weiterhin wurde Aminoethyl-Carbamoyl-ATP (EDA-ATP) als inverser Agonist an cyprinoiden Orthologen des GPR34 identifiziert. Die Erweiterung des möglichen Ligandenspektrums von Lipiden zu Nukleotidderivaten gibt Hinweise auf die Promiskuität der Bindungsstelle des GPR34. Die Ergebnisse zeigen, dass Lyso-PS nur eine zufällige Aktivität an einigen Orthologen des GPR34 hat. Mit Identifizierung eines Nichtlipides als invers-agonistischen Liganden ist die Suche nach dem natürlichen Liganden des GPR34 noch nicht abgeschlossen und sollte auf weitere chemische Entitäten ausgeweitet werden. / Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

Récemment plusieurs récepteurs couplés aux protéines G (RCPGs) ont été caractérisés au niveau des membranes intracellulaires, dont la membrane nucléaire. Notre objectif était de déterminer si les sous-types de récepteurs β-adrénergiques (βAR) et leurs machineries de signalisation étaient fonctionnels et localisés à la membrane nucléaire des cardiomyocytes. Nous avons démontré la présence des β1AR et β3AR, mais pas du β2AR à la membrane nucléaire de myocytes ventriculaires adultes par immunobuvardage, par microscopie confocale, et par des essais fonctionnels. De plus, certains partenaires de signalisation comme les protéines GαS, Gαi, l’adénylate cyclase II, et V/VI y étaient également localisés. Les sous-types de βAR nucléaires étaient fonctionnels puisqu'ils pouvaient lier leurs ligands et activer leurs effecteurs. En utilisant des noyaux isolés, nous avons observé que l'agoniste non-sélectif isoprotérénol (ISO), et que le BRL37344, un ligand sélectif du β3AR, stimulaient l'initiation de la synthèse de l’ARN, contrairement à l'agoniste sélectif du β1AR, le xamotérol. Cette synthèse était abolie par la toxine pertussique (PTX). Cependant, la stimulation des récepteurs nucléaires de type B de l’endothéline (ETB) causaient une réduction de l'initiation de la synthèse d’ARN. Les voies de signalisations impliquées dans la régulation de la synthèse d’ARN par les RCPGs ont ensuite été étudiées en utilisant des noyaux isolés stimulés par des agonistes en présence ou absence de différents inhibiteurs des voies MAP Kinases (proteines kinases activées par mitogènes) et de la voie PI3K/PKB. Les protéines impliquées dans les voies de signalisation de p38, JNK, ERK MAP Kinase et PKB étaient présents dans les noyaux isolés. L'inhibition de PKB par la triciribine, inhibait la synthèse d’ARN. Nous avons ensuite pu mettre en évidence par qPCR que la stimulation par l’ISO entrainait une augmentation du niveau d'ARNr 18S ainsi qu’une diminution de l'expression d’ARNm de NFκB. En contraste, l’ET-1 n’avait aucun effet sur le niveau d’expression de l’ARNr 18S. Nous avons ensuite montré que la stimulation par l’ISO réduisait l’expression de plusieurs gènes impliqués dans l'activation de NFκB, tandis que l’inhibition de ERK1/2 et PKB renversait cet effet. Un microarray global nous a ensuite permis de démontrer que les βARs et les ETRs nucléaires régulaient un grand nombre de gènes distincts. Finalement, les βARs et ETRs nucléaires augmentaient aussi une production de NO de noyaux isolés, ce qui pouvait être inhibée par le LNAME. Ces résultats ont été confirmés dans des cardiomyocytes intacts en utilisant des analogues cagés et perméables d’ISO et de l'ET-1: l'augmentation de NO nucléaire détectée par DAF2-DA, causée par l'ET-1 et l'ISO, pouvait être prévenue par le LNAME. Finalement, l’augmentation de l’initiation de la transcription induite par l'ISO était aussi bloquée par le L-NAME ou par un inbitheur de PKG, le KT5823, suggérant que la voie NO-GC-PKG est impliquée dans la régulation de la transcription par les βAR. En conclusion, les βARs et les ETRs nucléaires utilisent des voies de signalisation différentes et exercent ainsi des effets distincts sur l’expression des gènes cardiaques. Ils représentent donc une avenue intéressante pour le développement de drogues pharmacologiques. / Recently several G protein-coupled receptors (GPCRs) have been shown to localize to intracellular membranes, in particular the nuclear membrane. As such, we sought to determine if the β-adrenergic receptor (βAR) subtypes and their associated signalling machinery are functionally localized to nuclear membranes. We demonstrated the presence of β1AR and the β3AR, but not the β2AR, in adult ventricular myocyte nuclei by western blotting, confocal microscopy and functional assays. Downstream signalling partners such as GαS, Gαi and adenylyl cyclase II and V/VI were also present. Nuclear-localized βARs were functional with respect to ligand binding and effector activation. In isolated nuclei, the non-selective βAR agonist isoproterenol (ISO) and the β3AR-selective ligand BRL37344, but not the β1AR-selective xamoterol, stimulated transcription initiation in a pertussis toxin (PTX)-sensitive manner. In contrast, stimulation of type B endothelin receptors (ETB), another GPCR family shown to be present on the nuclear membrane, decreased de novo RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of the PI3K/PKB and mitogen-activated protein kinase (MAPK) pathways. Components of p38, JNK, and ERK1/2 MAPK cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine converted the activation of the βAR from stimulatory to inhibitory with regards to transcription initiation. Analysis by qPCR indicated isoproterenol treatment increased 18S rRNA but decreased NFκB mRNA. In contrast, ET-1 had no effect on 18S rRNA expression. Further investigation using pathway-specific PCR arrays revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB and that ERK1/2 and PKB inhibitors attenuated this effect. Subsequent genome-wide microarray analysis has revealed that nuclear βAR and ETB regulated a host of genes in an overlapping but distinct manner. Moreover, both ET-1 and ISO produced an L-NAME-sensitive increase in NO production in isolated cardiac nuclei. These observations were confirmed in intact cardiomyocytes using novel caged analogues of ISO and ET-1 and the cell-permable NO-sensitive fluorescent dye, DAF-2 DA. Briefly, both ET-1 and isoproterenol increased NO production, and this increase was prevented upon preincubation with L-NAME. Moreover, the ability of isoproterenol to increase transcription initiation in isolated nuclei was blocked by L-NAME or the PKG inhibitor KT5823, indicating the NO-GC-PKG pathway is involved in the regulation of gene expression by nuclear βARs. Hence, we have shown that βARs and ETRs in the nuclear membrane activate distinct signalling pathways, resulting in different effects on gene transcription and thus represent potentially important targets for drug development.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de protéines membranaires du génome humain. Ils transmettent les signaux extracellulaires provenant de plusieurs stimuli comme les odeurs, les ions, les hormones et les neurotransmetteurs, à l'intérieur des cellules. En se liant aux RCPGs, ces molécules contribuent à la stabilisation des changements conformationnels activateurs qui se propagent jusqu'au domaine intracellulaire des récepteurs. Ces derniers engagent ensuite un ou plusieurs effecteurs, comme les protéines G hétérotrimériques et les β-arrestines (βarrs), qui activent une cascade d'événements moléculaires menant à la réponse cellulaire.Récemment, la publication de structures cristallines de RCPGs liant des ligands diffusibles a offert une opportunité de raffiner à une résolution atomique les modèles des mécanismes de transduction des signaux. Dans la première partie de cette thèse, nous avons donc exploré les déterminants de la signalisation du récepteur prototypique β2-adrénergique (β2AR), induite par les β-bloqueurs. En ne tenant compte que de leur efficacités sur le β2AR dans les voies de l'adénylate cyclase (AC) et des protéines kinases activées par les facteurs mitogéniques (MAPK), les β-bloqueurs peuvent être classés en 3 groupes distincts (agoniste inverse AC / agoniste MAPK, antagoniste neutre AC / agoniste MAPK et agoniste inverse AC / agoniste inverse MAPK). Afin de déterminer le lien entre leur efficacité et leur mode de liaison, nous avons réalisé des expériences d'arrimages moléculaires in silico entre des β-bloqueurs de chacun des groupes et la structure cristalline du β2AR liée au carazolol. De manière intéressante, les ligands à l'intérieur d'un groupe partagent un mode de liaison, alors que ceux des ligands entre les groupes divergent, suggérant que le mode de liaison des β-bloqueurs pourrait être utilisé pour prédire leur l'efficacité. En accord avec cette hypothèse, nous avons prédit et confirmé l'efficacité agoniste MAPK du carazolol, un inverse agoniste AC du β2AR se liant au récepteur de manière similaire au groupe inverse agoniste AC / agoniste MAPK. De manière intéressante, le groupement aryl des ligands agonistes inverses agonistes AC / agoniste MAPK, le seul groupement chimique variable de ce groupe, est prédite pour lier la région des 3e et 5e hélices transmembranaires (TM3 et TM5). Nous avons donc émis l'hypothèse que cette région pourrait être un déterminant de l'efficacité de ces ligands. En accord avec cette dernière, la mutation de 2 résidus (T118I, S203A) localisés proches du site de liaison des groupements aryls des β-bloqueurs, prévient l'efficacité agoniste inverse de l'ICI-118551 sur la voie de l'AC sans affecter l'efficacité d'un agoniste, indiquant que cette région est importante pour la transmission de l'effet agoniste inverse, du moins sur la voie de l'AC. Les βarrs sont des protéines d'échafaudage qui coordonnent la formation de complexes avec plusieurs dizaines d'effecteurs de signalisation. Originalement identifiées pour leur rôle dans la désensibilisation et l'internalisation des RCPGs, elles sont aussi d'importants effecteurs de la signalisation des RCPGs indépendante des protéines G hétérotrimériques. Cependant, contrairement aux protéines G hétérotrimériques, il n'existe que peu d'outils pour les étudier. Ainsi, la deuxième partie de la thèse est dédiée au développement d'outils pour l'étude des βarrs. À cette fin, nous avons d'abord tenté de transposer une méthode de mesure de l'interaction entre 2 protéines par la technologie de transfert d'énergie de bioluminescence par résonance (BRET) en microscopie et chez des souris transgéniques afin de mesurer de manière subcellulaire et dans un contexte natif l'engagement de la βarr à des RCPGs. Ainsi, nous avons établi les preuves de principe que le BRET peut être utilisé pour localiser l'interaction entre la βarr et le récepteur de la vasopressine de type 2 (V2R) sur une cellule au microscope et pour détecter l'interaction entre la βarr et le β2AR sur des tissus de souris transgéniques exprimant ces protéines fusionnées avec des partenaires BRET. Finalement, il n'existe aucun inhibiteur pharmacologique ciblant les βarrs. Ainsi, grâce à la combinaison d'approches de criblage virtuel sur un modèle de la structure des βarrs et d'essais de validation cellulaire, nous avons développé un inhibiteur pharmacologique des βarrs. À l'aide de cet outil, nous avons confirmé l'implication des βarrs dans l'activation des MAPK par le V2R, mais aussi montré un nouveau rôle des βarrs dans le recyclage du β2AR. Les connaissances et outils développés dans cette thèse permettront de mieux comprendre les déterminants moléculaires de la signalisation des RCPGs et entre autres, grâce à des nouvelles approches pour étudier le rôle cellulaire et physiologique des βarrs. / G Protein-Coupled Receptors (GPCR) are members of the largest family of membrane protein in the human genome. They transduce the signal from a variety of stimuli like odors, ions, hormones and neurotransmitters, inside the cells. By binding directly to the receptors, these molecules stabilize activating conformational changes that are allosterically propagated through transmembrane to intracellular domains. Effectors like heterotrimeric G protein and β-arrestins (βarrs) are then engaged by activated receptors and trigger a cascade of signalling events leading to a cellular response. Recently, the resolution of the crystal structure of GPCR that bind to freely diffusible ligands provided the opportunity to refine at an atomic level the models describing the mecanisms of receptor signal transduction. In the first section of this thesis, we have explored the determinants of the prototypical β2-adrenergic receptor (β2AR) signalling induced by β-blockers. Given their efficacy on Adenylate Cyclase (AC) and Mitogen-Activated Protein Kinase (MAPK) pathways, β-blockers can be classified within 3 signalling groups (AC inverse agonist / MAPK agonist, AC neutral antagonist / MAPK agonist and inverse agonist for AC and MAPK). In order to gain insight on the relation between their efficacy and binding mode, we performed in silico binding experiments between β-blockers from each group and the β2AR crystal structure bound to carazolol. Interestingly, ligands within a group share similar binding mode in contrast to those of different groups, suggesting that β-blockers binding mode could be used to predict their efficacy. In accordance to this hypothesis, we have predicted and confirmed that carazolol, an AC inverse agonist that bind to β2AR in a similar way than the AC inverse agonist / MAPK agonist group, is indeed an agonist for MAPK pathway. Moreover, aryl chemical function from AC inverse agonist / MAPK agonist ligands, barely the only variable structure feature of this group, was predicted to bind β2AR nearby the transmembrane helices 3 and 5 (TM3 and TM5). We thus have predicted that this region would be a determinant of the AC inverse agonist / MAPK agonist ligand efficacy. Accordingly, we found that mutation of 2 residues (T118I, S203A) close to the aryl moiety binding site prevents inverse agonist efficacy of ICI-118551 on AC pathway, without affecting agonist efficacy, indicating that this receptor region is important for the efficacy of these group of β-blockers, at least on AC inverse agonism.βarrs are scaffolding proteins that coordinate protein complex formation with dozen of signalling effectors. First identified for their role on GPCR desensitization and internalization, βarrs are also an important heterotrimeric G protein independent GPCR signalling effectors. However, in contrast to heterotrimeric G protein, only a few tools are available for their study. Thus, the second section of this thesis aim at developing tools for the study of βarrs. For this purpose, we had attempted to transpose a method to measure protein-protein interaction that use Bioluminescence Resonance Energy Transfer (BRET) technology, in microscopy and in transgenic mice, in order to detect subcellular localization and in a native context the engagement of βarr to RCPGs. Thus, we have established a proof of principle that BRET can be combined with microscopy to locate an interaction between βarr and the type 2 vasopressin receptor (V2R) within a cell. Moreover, we have established a second proof of principle that we can detect βarrs recruitment to β2AR on cells extracted from tissues of transgenic mice expressing these proteins fused to BRET partner. Finally, there is no pharmacological inhibitor of βarrs. Thus, using a combination of virtual screening and cellular validation approches, we have developed the first pharmacological βarrs inhibitor. With this novel tool, we have confirmed the implication of βarrs in V2R-mediated MAPK activation, but also showed a new role of βarrs in β2AR recycling.The finding and the tools presented in this thesis should allow to better understand the molecular determinants of GPCR signalling, and among other things, by proposing new tools to study βarrs cellular and physiological roles.