The signaling pathways of tyrosine kinase Trk receptors and their downstream biological effects are well known, but the parameters controlling the interactions between the receptors and their natural ligands still remain to be defined. Recent published reports from our laboratory indicate that nerve growth factor (NGF)-induced TrkA receptor activation is dependent on a membrane cellular sialidase. This sialidase activity specifically targets and hydrolyzes sialyl α-2, 3-linked β-galactosyl residues resulting in the desialylation and activation of the receptor. These findings support a novel hypothesis that places mammalian sialidase(s) in a cycle of activation of these receptors by their natural ligand. Taken together, they also predict a prerequisite desialylation of Trk receptors caused by a sialidase on the cell surface enabling the removal of a steric hindrance to receptor dimerization. Until now, the sialidase associated with neurotrophin-treated live Trk-expressing cells has not been identified. The molecular mechanism(s) of sialidase activation by neurotrophin factors binding to their receptors also remains unknown. In this thesis, the novel role of Neu1 sialidase in the activation of ligand-induced TrkA and TrkB receptors has been identified. It has been reported for the first time that Neu1 is already in complex with naïve and ligand-induced Trk receptors. In addition, a membrane sialidase mechanism initiated by NGF binding to TrkA has been indentified. It suggests a potentiation of GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live TrkA- and TrkB-expressing cells and primary neurons. These results establish a unique mode of regulation of Trk receptors by their natural ligand and define a new function for Neu1 sialidase. Preliminary data indicate that members of the family of tyrosine kinase receptors like epidermal growth factor receptor (EGFR) and insulin receptor are also under the same regulatory control of Neu1 sialidase. Recent reports from the laboratory have indicated that ligand-induced activation of the highly glycosylated Toll-like receptors, TLR-2,-3 and -4 is also dependent on Neu1 sialidase on the cell surface. Taken all together, the findings in this thesis uncover a Neu1 and MMP-9 cross-talk on the cell surface which is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and neuron function. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-26 11:44:51.418

The Luteinizing hormone receptor (LHR) belongs to the G protein-coupled receptor family, asdo the other glycoprotein hormone receptors for FSH, TSH, and CG. The LHR comprises twohalves of ~350 amino acids: an extracellular hormone binding exodomain and a seventransmembrane-spanning endodomain responsible for signal generation. Hormone binds to theexodomain with high affinity, and the resulting conformational changes in thehormone/exodomain complex modulate the endodomain to generate hormone signals. Hormonebinding to an LHR produces hormonal signals (cis-activation), but it is not known whether aliganded LHR could activate other unoccupied LHRs (trans-activation). The LHR activates bothadenylyl cyclase and phospholipase C??. This dissertation shows that trans-activation of the LHRleads to the activation of adenylyl cyclase to induce cAMP but not to the activation ofphospholipase C?? to induce the inositol phosphate signaling. Trans-activation offers amechanism of signal amplification at the receptor level and also provides a mechanism ofmultiple signal generation for a liganded LHR to cis-activate phospholipase C?? and transactivateadenylyl cyclase. Also coexpression of Gi2 with a constitutively activating LHR(Asp578Gly), the most common mutation of male-limited precocious puberty, shows that Gi2could completely inhibit cAMP induction by the LHR mutant. Experiments using the carboxylterminal region of G protein ?? subunits demonstrate that LHR has overlapping binding sites forG?? subunits Gs and Gi2.

The aims of this thesis were (1) to learn about the identities of the molecules involved in the maintenance of long-term potentiation (LTP), and (2) to develop and test a behavioural paradigm capable of elucidating the interaction between these molecular processes and the persistence of long-term memories. By improving the stability of field recordings in in vitro electrophysiology, it was possible to investigate the molecular processes that determine the long-term changes in synaptic efficacy. In these experiments, the interactions between two convergent inputs onto the same neuronal population in the CA1 region of the hippocampus were monitored for over ten hours. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders, and test stimulation over long periods of time after LTP-induction, enabled a pharmacological dissociation of potentially distinct roles of the calmodulin kinase (CaMK) pathways in LTP. This places constraints on the mechanisms by which synaptic potentiation, and possibly memories, become stabilized. The experiments show that tag setting is blocked by the CaMK inhibitor KN-93 that, at low concentration primarily blocks CaMKII, whereas a CaMKK inhibitor, STO-609, selectively limits the synthesis or the availability of plasticity related proteins (PRPs). To test whether memories can be subject to modulation by independent experiences, behavioural studies tested the possibility of lengthening the persistence of a relatively weak memory by pairing its induction with an event capable of inducing the synthesis of the required PRPs. Corticosterone-dependent stressful events like a cold swim proved to interfere and weaken spatial memories. On the other hand, the exploration of a novel environment succeeded in rescuing the decay of a weak memory. The effect of the exploration of the novel environment was dependent on NMDA and dopamine receptor activation, as well as protein synthesis. These results are discussed in relation to the synaptic tagging and capture hypothesis and a novel model of the neuronal mechanisms underlying synaptic plasticity is developed from them.

Myocardial infarction (MI) occurs following acute thrombotic occlusion of a coronary artery, and triggers a robust inflammatory response. Within hours, neutrophils are recruited to the infarcted myocardium followed by the infiltration of pro-inflammatory Ly6Chi monocytes. Transition from the pro-inflammatory macrophage phenotype (M1) to an anti-inflammatory, pro-resolution phenotype (M2-like) is critical to successful infarct healing. Interventions that polarize macrophages towards an anti-inflammatory 'M2-like' phenotype improve infarct healing in the experimental MI mouse model and reduce subsequent adverse remodelling of the myocardium, but the endogenous mechanisms that regulate repair are not well understood. Furthermore, differences in the resolution of inflammation in C57BL/6 and BALB/c mice, which are two of the commonly used wild-type mouse strains in experimental MI have not been characterised. We previously found that low peripheral blood eosinophil count is associated with increased short-term risk of mortality in low-intermediate risk patients with ischaemic heart disease. This suggests that eosinophils may have a role in the successful remodelling and repair of the heart following myocardial infarction. Eosinophils express a number of immuno-modulating cytokines and lipid mediators implicated in the resolution of inflammation. Increasingly prominent is interleukin-4 (IL-4), a cytokine that has been found to maintain the anti-inflammatory M2-like phenotype in macrophages. We therefore hypothesised that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are key in regulating the subsequent inflammatory response and scar tissue formation during infarct repair and cardiac remodelling. Experimental MI was induced by permanent left anterior descending artery ligation in isofluorane anaesthetized 12-15 week-old male wild-type (WT) BALB/c, WT C57BL/6, IL4Rα-/-, IL-4Rαflox/-, IL-4Rαflox/-LysMCre mice and eosinophil-deficient ΔdblGATA mice. Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of single cell infarct and remote zone tissue digests. Blood eosinophil count and 6-month all-cause mortality were assessed in 732 consecutive patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). The rate of mortality due to cardiac rupture was significantly higher in C57BL/6 mice in comparison with BALB/c mice at Day 7 post-MI. This was associated with a higher proportion of pro-inflammatory Ly-6Chi macrophages infiltrating the infarct zone tissue of C57BL/6 mice following MI. An accompanying reduction in the number of splenic Ly-6Chi monocytes post-MI, suggestive of splenic monocyte mobilisation, was seen in C57BL/6 mice but not found in BALB/c mice. Furthermore, C57BL/6 mice had a delayed transition in macrophage polarisation towards an anti-inflammatory phenotype. Disruption of IL4Rα signalling, in mice null for the IL4Rα gene, resulted in increased F4/80+ macrophage and pro-inflammatory Ly6Chi macrophage infiltration of the infarct zone and reduced expression of the anti-inflammatory macrophage marker CD206, compared to wild-type controls. Furthermore, expression of GATA3 and ST2, both associated with the immunosuppressive function of (CD4+ Foxp3+) regulatory T cells, was reduced in infarct zone regulatory T cells from IL4Rα-/- mice. These findings were associated with defective wound healing with impaired angiogenesis, increased scar size, disarrayed infarct zone collagen deposition, accompanied by modified expression of plod2 that encodes the collagen cross-linking enzyme lysyl hydroxylase 2. Resulting in greater left ventricular dilatation and loss of cardiac function, as well as a higher 7- day mortality due to cardiac rupture in IL4Rα-/- mice. This indicates that successful infarct repair requires the engagement of IL-4Rα signalling to facilitate the accumulation of anti-inflammatory macrophages and highly immunosuppressive ST2+ regulatory T cells in the heart following MI. Resident cardiac macrophages from naïve hearts of IL-4Rαflox/-LysMCre mice failed to undergo LysMCre-mediated deletion of the IL-4Rα gene, potentially because low or absent expression of Lyz2 (encoding lysozyme M). In both ST-elevation MI (STEMI) patients and mice after acute MI, there was a decline in peripheral blood eosinophil count, with activated eosinophils being recruited to the infarct zone and paracardial adipose tissue of mice. The transcription factors GATA-1 plays a role in the differentiation of eosinophils from eosinophil progenitor cells. Deletion of GATA-1 results in loss of the eosinophil lineage and has been exploited to develop the eosinophil-deficient ΔdblGATA mouse. ΔdblGATA mice were used to address the role of eosinophils in cardiac remodelling following MI. ΔdblGATA mice had increased left ventricular dilatation and reduced ejection fraction after induction of MI, relative to wild-type mice. ΔdblGATA mice had increased scar size with disarrayed infarct zone collagen deposition, accompanied by modified expression of the genes plod2 and lox, which are associated with collagen cross-linking. The proportion of CD206+ anti-inflammatory macrophages was less in the infarct zone of ΔdblGATA mice, but was restored by adoptive transfer of eosinophils from WT mice. Furthermore, adverse cardiac remodelling in eosinophil-deficient ΔdblGATA mice was rescued by provision of IL-4 complex following MI. In conclusion, an enhanced inflammatory response following MI underlies the increased risk of cardiac rupture seen with WT C57BL/6 mice in comparison to WT BALB/c mice. WT BALB/c mice are protected from cardiac rupture, which was associated with an absence of splenic monocyte mobilisation following ischaemic injury. The resolution of inflammation was found to be dependent on IL4Rα signalling which is crucial for cardiac repair and remodelling, through modulating inflammatory cell recruitment and phenotype, as well as scar formation. Eosinophils are recruited to the heart post-MI and are essential for regulating cardiac repair and remodelling, likely through provision of IL-4. Therefore, we were able to show that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are both key in regulating the subsequent inflammatory response and scar tissue formation during infarct healing and cardiac remodelling.

No description available.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of β integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin β1 to focal adhesions and enhances integrin β1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

It has been shown that the amino terminus and second extracellular loop (EC2) of CXCR2 are crucial for ligand binding and receptor activation. The lack of an ionic lock motif in the third intracellular loop of CXCR2 focuses an investigation of the mechanism by which these two extracellular regions contribute to receptor recognition and activation. The first objective of this investigation was to predict the structure of CXCR2 based on known structures of crystallized GPCRs. Rhodopsin, β2-adrenergic receptor, CXCR4 were used for homology modeling of CXCR2 structure. Highly conserved motifs found in sequence alignments of the template GPCRs were helpful to generate CXCR2 models. We also studied solvent accessibility of residues in the EC2 of CXCR2 in the inactive state. Most of the residues in the EC2 were found to be solvent accessible in the inactive state, suggesting the residues might be involved in ligand recognition. Second, we studied the role of charged residues in the EC2 of CXCR2 in ligand binding and receptor activation using constitutively active mutants (CAM) of CXCR2, D9K and D9R. Combinatorial mutations consisting of the CAM in the amino terminus and single mutations of charged residues in the EC2 were generated to study two concepts including “attraction” and “repulsion” models. The mutant receptors were used to test their effects on cell surface expression, ligand binding, receptor activation through PLC-β3, and cellular transformation. All the mutations in the repulsion model result in CXCR2 receptors that are unable to bind ligand, suggesting that each of the Arg residues in the EC2 are important for ligand recognition. Interestingly, mutations in the attraction model partially inhibited receptor activation by the CAM D9K, suggesting that Glu198 and Asp199 residues in the EC2 are associated with receptor activation. Furthermore, a novel CAM, E198A/D199A, was identified in this study. These negatively charged residues are very close to a conserved disulfide bond linking the EC2 and the third transmembrane. In this sense, these current discoveries concerning the structural basis of CXCR2 and interdisciplinary approaches would provide new insights to investigate unknown mechanisms of interaction with its cognate ligands and receptor activation.

GPCRs, influencing myriads of cellular functions, are the members of the largest family of the membrane proteins. However, their structures and the signaling mechanisms still remain enigmatic. In case of the Glycoprotein Hormone Receptor (GpHR) family the structure-function relationship is less understood because of a large extra-cellular domain (ECD). This large ECD, consisting of Leucine Rich Repeats (LRRs) and membrane-proximal hinge region, is sufficient for specific binding to the hormone (Ascoli, Fanelli, & Segaloff, 2002), but for receptor activation, hormone binding is translated via a conformation wave starting at hinge region and relayed to the transmembrane domain. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure-function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005; Fox, Dias, & Van Roey, 2001; Wu, Lustbader, Liu, Canfield, & Hendrickson, 1994). However, the complete understanding of the hormone‐receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action or regulating receptor activation in a hormone independent manner. The crystal structure of FSHR ECD (amino acids 17-366) revealed that LRRs form a semicircular palm shaped structure with the C terminus region, designated as the hinge region, protruding out like a thumb. The hinge region, rather than being a separate functional unit, was found to be an integral part of the LRR domain, having two such repeats (LRR11 &12). LRR 11 is connected to LRR12 through a hairpin loop (amino acids 280-344) harboring the invariant sulfated tyrosine residue (sTyr) in YD/EY motif (X. Jiang et al., 2012). The heterodimeric hormones consisting of a common  subunit and a hormone specific  subunit, bind to the primary hormone binding site at LRR 4-6 as reported in the FSHR-FSH co crystal (Fan & Hendrickson, 2005). This primary binding of the hormone at LRR 4-6 creates a pocket (comprising of the residues P16α, L17α, F18α, F74α, L37β, Y39β, and P45β) in the hormone for secondary binding at sTyr residue. This interaction is proposed to initiate conformation change in the hinge region which further leads to FSHR activation (X. Jiang et al., 2012). Thus, the role of hinge region in GpHR activation got evolved from a linker to a switch, which decides the fate of the receptor activity (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). sTyr residue being conserved, presents itself as a potential player in activation mechanism of all the three receptors of the family (Bonomi, Busnelli, Persani, Vassart, & Costagliola, 2006; Kreuchwig, Kleinau, & Krause, 2013). Precise involvement of sTyr in GpHR activation is yet to be explored. The previous studies from the laboratory using the hinge region specific polyclonal and monoclonal antibodies established the unequivocal role of the hinge region in FSHR and TSHR activation (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). However, its function in LHR activation has not been conclusively established. Due to the unavailability of the structural information of LHR ECD/hinge, it is more difficult to study and explain the role of hinge region in LHR activation. The hormone independent signaling by point mutants of LHR also remains poorly understood. In the present study an attempt has been made to understand the role of the hinge region in LHR signaling and modulating role of LRRs in hinge mediated LHR activation. The present study was initiated with an overall objective of understanding the molecular details of LHR activation mechanism keeping hinge at the centre of the picture. To have clarity of this picture with a holistic view of the mechanism, multi-pronged approach was adopted. Initially, ScFvs against LHR hinge region were employed as tools to probe into the hormone‐receptor interactions. Antibodies against glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone‐receptor interactions and signal transduction (Agrawal & Dighe, 2009; Dighe & Moudgal, 1983; Gadkari, Sandhya, Sowdhamini, & Dighe, 2007; Gadkari et al., 2007; Kene, Nalavadi, Dighe, Iyer, & Mahale, 2004; Majumdar, Railkar, & Dighe, 2012a, 2012b). In this study, Single chain Fragment variables (ScFvs) against the hinge region of LH receptor have been employed to understand the mechanism of receptor activation. The effects of LHR ScFvs on hCG-LHR interactions have been investigated and three of the ScFvs, JE10, JE4 and JG1 could bypass the hormone and activate the receptor directly, with JE10 being the most potent one. The effect on the signaling was specific for LHR as no increase in cAMP response was observed for TSHR/FSHR in presence of these ScFvs. JE10 surprisingly was unique and could alter the hCG-LHR interaction by decreasing hormone affinity and simultaneously increasing the Bmax for the hormone. JE10 binding was decreased to the pre-formed hormone receptor complex suggesting that hCG and the stimulatory antibody show stearic hindrance at the binding sites on hinge or hormone binding induces conformational change in the epitope of JE10. The change in affinity and Bmax of the hormone by JE10 could be due to unmasking of new binding sites for hormones or an allosteric effect on the protomer interaction like explained in case of a small TMD specific allosteric modulator of FSHR (Xuliang Jiang et al., 2014). JE10 could also potentiate hCG signaling at sub-saturating concentrations of hCG, the precise mechanism of which is not clear. Through TSHR-LHR chimeric mutants, a stretch from amino acids 313-349, within the hinge region, was identified as the site recognized by JE10. In order to study structural features of the JE10 epitope, LHR ECD was modeled on the basis of FSHRED crystal structure. With most of the motifs being structurally conserved (CF3 and YPSHCCAFF); the major portion of the hinge region was found to be unstructured. This unstructured region harbored the JE10 epitope as well as the functionally important conserved sTyr residue. The CD spectra of LHR hinge in presence of ScFv JE10 suggested a ScFv induced helical conformation and stabilization of the hinge loop region, which was constrained in the homology model into helices. As loop was now constrained in the Mode 2, so was the interaction of sTyr, which was now in contact with positively charged residues, probably stabilizing its charge. The YEY motif mutants further confirmed the indirect essential role of Y331 in activation of LHR by JE10. Another approach followed to study hCG-LHR interactions was use of a series of LHR N-terminal truncation mutants and truncation mutants along with one of the LHR CAM (S277Q/D578Y). The effect of these truncations on hormone binding and receptor activation was investigated. The deletion of Cysteine box (Cb-1) of LHR (present at N-terminus of ECD) leads to abrogation of hCG binding, indicating importance of this region in maintaining ECD conformation required for hormone binding. This is the most unexplored region of the ECD. Though Cb-1 does not bind to the hormone directly (as is evident from the crystal structure) but it is indirectly essential for hormone binding. The basal activity of these truncated mutants was as low as that of the wild type LHR, reconfirming that no region of LHR ECD acts as an inverse agonist for the TMD (Karges, Gidenne, Aumas, Kelly, & Milgrom, 2005). Truncation mutants with CAM (double mutants) also showed low basal activity, suggesting that intact ECD is prerequisite for keeping LHR in a conformation, best suited for hormone binding and binding of G protein for activation. That best conformation still needs to be explored. Truncation mutants did not get stimulated by JE10 also. This observation is opposite to the previous studies in which FSHR/TSHR truncated mutants could be stimulated by hinge specific antibodies (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). This difference points out to the variations in which LHR hinge-TMD interactions prevail and lead to the receptor activation. This variation was also confirmed with a previous report in which the binding of TSHR-ECL specific antisera to wild type LHR and TSHR-LHR 6 chimeric mutant suggested that hinge of LHR does not seem to be constraining the TMD (Majumdar et al., 2012b). Thus the LHR TMD itself possesses all the inhibitory interactions, also indicated by the presence of most of the activating mutations in LHR TMD (Piersma, Verhoef-post, Berns, & Themmen, 2007). Protomer interaction is the newest aspect of GpHR activation mechanism and has not reached any conclusive, physiologically relevant explanations yet. By co-transfection of wild type LHR and ECD truncated mutants, this study suggests the LHR protomer interaction and proposes the involvement of allosteric effect of ECD on LHR protomer interaction. The effect of JE10 on activating and inactivating mutants of LHR were quite interesting. The ScFv could bind to the activating mutant D578Y (associated with precocious puberty). This mutant exhibited higher basal cAMP production, but was activated even further by the ScFv. The inactivating mutant A593P is a completely inactive receptor associated with (associated with pseudo-hermaphroditism. It does not respond to the hormone at all. The ScFv JE10 binds to this receptor and stimulates cAMP production. This observation is rather striking, as it is possible to activate a completely inactive mutant that could not be stimulated by the hormone by a binder specific for the hinge region. It is not clear how the binder that interacts with the hinge region affects the function of the inactive TMD thus providing an interesting tool to investigate the interactions between the hinge region and TMD that are probably key to understand the activation of GpHR. which has been shown to be central to the GpHR activation mechanism, (Agrawal & Dighe, 2009; Majumdar et al., 2012b; Schaarschmidt, Huth, Meier, Paschke, & Jaeschke, 2014). As per the recently suggested model by Deupi et. al., that each mutation and agonist can take a different pathway during activation (Kobilka & Deupi, 2007). The activated state induced by JE10 in D578Y and A593P seems to be different from the wild type LHR, with each activated receptor state having different capacity to bind to the G protein. The difference in G protein capacity in itself reflects the different receptor turnover or different Gs uncouplings or different Gs binding affinities, which needs to be further investigated, opening up another avenue for exploration. There is a lacuna in understanding the signal relay from the hinge to TMD. However, JE10 seems to be activating the wild type LHR and the mutants directly or indirectly by modulating the 6th helix of the TMD, known to be important for hormone independent activation of LHR (Fanelli, 2000; Latronico & Segaloff, 2007; Majumdar et al., 2012b). As evident from the absence of any hinge mediated constrain on LHR TMD and absence of uncharged residues present in LHR LRRD-TMD interface (LHR ECD Model 1), LHR hinge does not seem to be maintaining significant interactions with the TMD in absence of a ligand or in its basal state. Hormone/ agonist binding or activating mutations act as a positive regulator (inducing conformation change in hinge), required to bridge the interactions between LHR hinge and the TMD, which is supported by various studies in the past (Karges et al., 2005; Majumdar et al., 2012b; Nishi, Nakabayashi, Kobilka, & Hsueh, 2002; Osuga et al., 1997; Ryu, Gilchrist, Tung, Ji, & Ji, 1998; Zeng, Phang, Song, Ji, & Ji, 2001). This interaction bridged by the conformational change in the hinge region, seems to isomerize the closed state of LHR into an activated state. The present study supports the conformational induction model for receptor activation in which intramolecular interactions between the two domains (hinge-TMD) lead to the receptor activation. In conclusion, this study presents a possible mechanism of activation of LHR by a partial agonist ScFv, which induces the conformation change in the disordered loop region (a.a.313-349) of the hinge and stabilizes it into helical state. This conformation change is predicted to be important for relaying the activation signal to the TMD. The study also demonstrates the activation of a completely inactive mutant A593P by JE10, suggesting a distinct possibility of its use as a therapeutic tool in treating infertility caused by inactivating mutations in LHR. On a second note, the study extends the role of LRRs, apart from direct hormone binding, to an indirect allosteric role in hormone binding, LHR activation and functional stability. This functional stability does not seem to be restricted to a single LHR but also depends on its interaction with nearby protomers. Though there are evidences for and against each of the above discussed possibilities, as yet there is no accepted model that explains the precise steps of receptor activation, hence, the molecular details of these interactions needs to be investigated in future.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19399号 / 医博第4050号 / 新制||医||1012(附属図書館) / 32424 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 川口 義弥, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Notch receptors and ligands are single-pass transmembrane proteins which play important roles in cell-cell communication. Notch in ‘harmony’ with other signaling pathways regulate the entire diversity of metazoan life (Artavanis-Tsakonas & Muskavitch, 2010). These signaling pathways also play key roles in regulatingseveral developmental processes. Given the importance of Notch signaling in various developmental decisions, it is not surprising that aberrant gain or loss-of-function of Notch pathway leads to several human diseases including cancer (Ranganathan et al, 2011). Notch signaling has also been implicated in various human cancers, most notably in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al, 2004). In view of the importance of Notch signaling in cancers, therapeutic molecules targeting this pathway are making their way into clinical trials (Rizzo et al, 2008). This underscores the importance of understanding the mechanism of Notch receptor activation in normal and patho-physiological conditions. In this thesis, antibodies against different domains of human Notch1 receptor have been used as tools to understand the mechanism of receptor activation. This work has provided insights into the role of Notch1 extracellular domain in ligand-dependent receptor activation. Further, the mechanism of ligand-independent receptor activation in T-ALL associated mutant Notch1 has also been investigated. This understanding of ligand-dependent and independent receptor activation facilitated development of mechanistic inhibitors of Notch signaling for therapeutic targeting of the cancer stem cells (CSCs) across the pectrum of cancers. The thesis is divived into two parts. Part-I focuses on understanding the role of Notch1 extracellular domain in receptor-ligand interactions using antibodies as a tool. In part-II, implications of these antibodies in therapeutic targeting of CSCs has been investigated. Part-I Unfolding the Mechanism of Notch1 Receptor Activation The extracellular domain of Notch1 receptor consists of 36 EGF-like repeats that contribute to ligand binding (Kopan & Ilagan, 2009). Despite extensive studies on the downstream consequences of Notch signaling, the initial events of ligandreceptor interactions have not been clearly elucidated. In the absence of structural insights into the receptor-ligand interactions, it was important to decipher the roles of various receptor domains in ligand-binding and consequent signaling. In this study, antibodies have been employed as tools for in-depth analyses of Notch receptorligand, interactions. Studies in Drosophila Notch receptor suggest that EGF-like repeats 11-12 are necessary and sufficient for ligand binding (Rebay et al, 1991). However, the role of these repeats in human Notch1 receptor-ligand interaction(s) was not clearly elucidated. Antibodies were generated against Notch1 EGF-like repeats 11-15. Further, these antibodies were characterized for their specificity for Notch1 receptor in various ligand-binding and signaling assays. The results suggest that the monoclonal antibodies (MAbs) against EGF-like repeats 11-12 were more potent inhibitors of ligand-binding compared to the antibodies against EGF-like repeats 13-15. As a part of these investigations, the Notch ligands Jagged1 and Jagged2, Delta-like1 and Delta-like4 were purified and characterized in various assays. Ability of these ligands to interact with Notch1 EGF-like repeat 11-15 was determined using Surface Plasmon Resonance. The Jagged family of ligands demonstrated higher affinity for this recept or fragment when compared to the Delta family of ligands. The relatively low affinities (μM) of all the ligands suggested possibile involvement of other EGF-like repeats in ligand-binding. This was further investigated using antibodies against other EGF-like repeats of Notch1. In Drosophila Notch EGF-like repeats 24-29 have been implicated in the ligand-dependent gain-of-function phenotype, suggesting a plausible involvement of this region in receptor activation (Pei & Baker, 2008). Therefore, role of human Notch1 EGF-like repeats 21-30 in ligand-binding and signaling was investigated. These EGF-like repeats demonstrated specific interaction with the ligand-binding domain (EGF-like repeats 11-15). This suggested that in the absence of the ligand, these inter-domain interactions keep the receptor in an auto-inhibited conformation. Further, ligand binding to EGF-like repeats 11-15 dissociated pre-formed interdomain interactions. These results suggested that, the binding of ligand to EGF-like repeat 11-12 overcomes the negative constraint imposed by the intra-domain interactions which might lead to receptor activation. Next, to understand the role of EGF-like repeats 21-30 in ligand binding, polyclonal antibodies were generated against the same and extensively characterized in various solid-phase and cell-based assays. These antibodies demonstrated partial inhibition of ligand-binding. Further, using immunoaffinity purified antibodies it was demonstrated that antibodies against EGF-like repeats 25-26 were most potent inhibitors of ligand-binding compared to antibodies against EGF-like repeats 21-24 and 27-30. These results provided novel insights into Notch1 receptor activation. The model proposed on the basis of these results suggested that ligand-binding to EGF-like repeats 11-12 competes with the inter-domain interaction, in turn dissociating EGF-like repeats 21-30 from the ligandbinding domain. It emerged that this altered conformation of the receptor creates a secondary ligand-binding site at EFG-like repeats 25-26. Overall these results provided novel insight into the mechanism of Notch receptor-ligand interaction(s). Part-II Implication in Cancer Stem Cell Targeting Recent studies have suggested existence of the CSC population in various cancers (Clevers, 2011). Notch signaling plays an important role in maintenance of these CSCs (Pannuti et al, 2010). Thus, targeting Notch signaling may provide a potential therapeutic tool for CSC targeting. Several studies have indicated that Notch1 receptor and ligands are overexpressed in breast cancer cells compared to the normal breast epithelium (Mittal et al, 2009; Reedijk et al, 2005; Reedijk et al, 2008). Moreover, it has been suggested that Notch1 signaling plays a key role in breast carcinogenesis (Stylianou et al, 2006). Monoclonal antibodies (MAbs) were used as mechanistic inhibitors of aberrant Notch1 signaling for therapeutic targeting of CSCs. One such antibody, MAb 602.101, against Notch1 ligand-binding domain (EGF-like repeat 11-12) inhibited proliferation and depleted breast CSCs. This MAb also modulated genes associated with stemness and epithelial to mesenchymal transition (EMT). Furthermore, MAb 602.101 irreversibly inhibited the sphere-forming potential of breast cancer cells by modulating long-term self renewing capacity of breast CSCs. Inhibition of Notch1 signaling by the MAb also depleted the chemoresistant CD44Hi/CD24Low sub-population in breast cancer cells. Interestingly, antibody treatment led to elevated expression of genes associated with myoepithelial lineage, which suggested that inhibition of Notch1 signaling might induce a differentiation program leading to reduction in the CSC population. This study demonstrated the importance of Notch1 signaling in CSCs and effectiveness of antibodies as a tool for specific targeting of individual Notch receptors in cancer therapeutics. While aberrant expression of receptors and ligands leads to breast cancer (Reedijk et al, 2005), gain-of-function mutations are associated with 40-50% of TALL\ patients (Weng et al, 2004). These mutations lead to ligand-independent receptor activation (Malecki et al, 2006). Despite several attempts of successful antibodymediated therapeutic targeting of Notch1 (Aste-Amézaga et al, 2010; Wu et al, 2010), specific antibodies recognizing T-ALL associated mutant Notch1 remains elusive. Using homology modeling, the mutation induced conformational change in T-ALL associated mutant Notch1 was predicted. These results suggested that mutation led to conformational changes in the Notch1 negative regulatory region (NRR) This conformation change might result in the constitutive activation of Notch1 signaling leading to pathogenesis. Next, MAbs were generated against the wild-type Notch1 NRR and characterized in flow-cytometry based assays for identification of conformation specific antibodies. These antibodies were classified as either wild-type specific, mutant specific or unbiased to receptor conformations. One such mutant specific MAb 604.107 demonstrated higher binding to mutant Notch1 in flowcytometer and SPR based experiments. This MAb also demonstrated specific inhibition of T-ALL associated mutant Notch1 signaling without affecting the wildtype signaling. Moreover, antibody treatment also inhibited proliferation and depleted leukemia initiating sub-population in patient derived T-ALL cells. Taken together, this study provides a novel tool for specific targeting of mutant Notch1 receptors in TALL. CSCs are inherently chemo-resistant and lead to tumor relapse (Chen et al, 2012). Recent studies have demonstrated a strong correlation between Notch1 signaling in lung CSCs and chemotherapy resistance (Hassan et al, 2013). In this study, Notch1 heterogeneity in solid tumors viz. breast and colon cancers was investigated. Using the antibodies generated previously in this study, Notch1High and Notch1Low sub-populations from MDA-MB-231 (breast cancer) and HCT-116 (colon cancer) cell lines were flow-sorted. It was demonstrated that the Notch1High subpopulation represented the sphere-forming CSCs in breast and colon cancer. The Notch1High sub-population also demonstrated chemo-resistant properties and expressed higher level of EMT and stemness markers. These results suggested explicit involvement of Notch1 signaling in EMT and maintenance of CSCs subpopulation in these cancers. The anti-Notch1 MAb also inhibited proliferation of the chemo-resistant Notch1High sub-population. Further, treatment with MAb inhibited expression of ABCC1 transporters in these drug-resistant cells leading to augmentation of chemotherapeutic response. Using mouse xenograft assays, it was demonstrated that Notch1 signaling plays an important role in the maintenacne of tumor-initiating sub-population in breast and colon cancer cells. Prior exposure of breast and colon cancer cells to MAb inhibited the tumor forming potential of these cells in xenotransplantation assays. Treatment with MAb alone or in combination with chemotherapy led to regression of pre-formed tumors in breast and colon xenograft models. These results demonstrated existence of Notch1 heterogeneity in breast and colon cancer cells and emphasised the importance of targeting Notch1 signaling to overcome drug-resistance in these cancers. The results described above have provided important insights into Notch1 receptor activation and this understanding was translated into therapeutic targeting of CSCs. This “proof-of-principle” demonstration has significant mechanistic and applied implications in Notch and cancer biology.