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by Anskar Yu-hung Leung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 147-163). / Chapter Section I --- Literature review / Chapter Chapter I.1. --- The epididymis - its structures and functions --- p.1 / Chapter Chapter I.2. --- Cellular mechanisms of transepithelial electrolyte transport in epididymis and other exocrine tissues --- p.5 / Chapter Chapter I.3 --- Signal transduction mechanism of chloride secretion in epididymis and other exocrine tissue --- p.11 / Chapter Chapter I.4 --- Significance of chloride secretion by the epididymal epithelium and the objectives of the study --- p.17 / Chapter Section II --- General methods / Chapter Chapter II.1. --- Tissue culture from the rat cauda epididymis --- p.20 / Chapter Chapter II.2. --- The short-circuit current technique --- p.30 / Chapter Chapter II.3. --- The immunofluorescence technique --- p.39 / Chapter Chapter II.4. --- Intracellular adenosine 3':5' cyclic monophosphate (cAMP) measurement --- p.43 / Chapter Chapter II.5 --- Intracellular Ca2+ measurement using the microfluorimetric technique --- p.49 / Chapter Section III --- Results / Chapter Chapter III.1. --- Studies on the neural and humoral controls of eletrogenic chloride secretion in rat epididymis --- p.55 / Chapter Chapter III.2. --- Local control of electrogenic chloride secretion in rat epididymis 226}0ؤThe role of the calcitonin gene-related peptide --- p.80 / Chapter Chapter III.3. --- Characterization of intracellular Ca2+ store using ATP as the calcium mobilizing agonist --- p.94 / Chapter Chapter III.4. --- Ca2+ handling mechanisms in single cultured rat epididymal cells --- p.106 / Chapter Chapter III.5. --- Studies on the effector process in the stimulus-secretion coupling- characterization of apical Cl- conductance in cultured rat cauda epididmal cells --- p.114 / Chapter Chapter III.6. --- Effects of secretory agonists on transepithelial Cl- transport and intracellular Ca2+ concentration in cultured human epididymal epithelium --- p.132 / Chapter Section IV --- General discussion --- p.142 / Chapter Section V --- References --- p.147 / Appendix --- p.164

Intracellular signaling cascades can no longer be viewed as linear pathways that relay and amplify information. Often, components of different pathways interact, resulting in signaling networks. The interactions of different pathways and the dynamic modulation of the activities of the components within signaling pathways can create a multitude of biological outputs. The cell appears to use these pathways as a way of integrating multiple inputs to shape a uniquely defined output. These outputs allow the cell to respond to and adapt to an ever-changing environment. Understanding how biological systems receive, process and respond to complex data inputs has important implications for the design and utilization of sensors for a variety of applications, including toxicology, pharmacology, medical diagnostics, and environmental monitoring. This study uses the elicitor sets method, which is an experimental framework designed to monitor information flows through signal transduction pathways. The elicitor set approach has been used to derive mechanistic interpretations from the action of Phenylmethylsulfonyl Fluoride (PMSF), a serine protease inhibitor and nerve agent analog. The elicitor panel comprises of signal transduction network effectors namely forskolin, clonidine, cirazoline and H89, each of which targets the signaling pathway at known specific points. The elicitor set experiments enable compartmentalization of the cAMP signaling pathway, examining the role played by each segment and identifying possible cross-talk mechanisms. Our experiments substantiate that selection of adenyl cyclase as the reference node and 10 [mu]M forskolin as the primary elicitor, segments the upper portion of the G-Protein Coupled Receptor (GPCR) pathway associated with the G[sub q] and G[sub i] proteins. Application of the secondary elicitors, 100 nM clonidine (a2-adrenergic receptor agonist), 1 pM and 100 pM cirazoline (al-adrenergic receptor agonists), and 1 [mu]M and 100 [mu]M H-89 (PKA inhibitor) fortifies the decoupling, as the system is unresponsive to clonidine and cirazoline in the presence of forskolin, while continuing to respond to H-89. Exposure of the cells to 1 mM PMSF subsequent to forskolin addition restricted the quantifiable impact of PMSF to regions of the signaling pathways below adenyl cyclase. Triggering the system by use of secondary elicitors augmented the information resolution which is reinforced by the increased sensitivity of cells to 100 [mu]M H-89 that acts at an important checkpoint below adenyl cyclase. / Graduation date: 2004

Summary : Macropinocytosis refers to the formation of large primary endocytic vesicles of irregular size and shape, generated by actin-driven evaginations of the plasma membrane, whereby cells avidly incorporate extracellular solutes. Macropinosomes resemble "empty" phagosomes and show no difference with the "spacious phagosomes" triggered by the enteropathogenic bacteria Salmonella and Shigella. Macropinosomes are formed at the leading edge and appear tightly regulated. They may fuse with lysosomes or regurgitate their content back to the extracellular space. Transformation of fibroblasts by Src or Ras results into the constitutive formation of macropinosomes at "ruffling" zones. My thesis adresses the dynamic of the constitutive macropinocytosis and its potential regulators. The first part of the thesis deals with the fate of macropinosomes. We found that, in v-Src transformed fibroblasts, macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase, forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer, horseradish peroxidase, in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phosphatidylinositol 3-kinase (PI3K) inhibitors and phosphatidylinositol-specific phospholipase C (PI-PLC), dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of peroxidase entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited its accumulation only after intervals >5 minutes, due to accelerated regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom. The second part of my thesis deals with the regulation of macropinocytosis. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection with an expression vector for wild-type PI3K regulatory subunit p85a, acting as dominant-positive PI3K, constitutively lead to stress fibers disruption, cortical actin recruitment, extensive ruffling and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and PI-PLC, as assayed by 3-phosphoinositides synthesis in situ and in vitro and IP3 steady state levels, respectively, and were abolished by stable transfection of Src-transformed cells with an expression vector for truncated p85a, acting as dominant-negative PI3K, as well as by pharmacological inhibitors of PI3K and PI-PLC, indicating requirement for both enzymes. These inhibitors strongly decreased the population of cells showing active membrane ruffling and did not detectably affect receptor-mediated endocytosis of transferrin. Interestingly, the effect of Src and dominant-positive p85a transfection on the actin cytoskeleton could be reversed within 30-60 min by the pharmacological inhibitor of PI3K, wortmannin. Whereas PI3K activation resisted PI-PLC inhibition by NCDC, PI-PLC activation was abolished by the PI3K inhibitor wortmannin and upon dominant-negative PI3K transfection, thus placing PI-PLC downstream of PI3K. Taken together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of actin cytoskeleton in oncogene-transformed fibroblasts resulting into constitutive ruffling and macropinocytosis.Résumé : La macropinocytose désigne la formation de grandes vésicules endocytaires primaires de taille et de forme irrégulière, produites par évagination de la membrane péricellulaire, qui permet l'accumulation accélérée de solutés extracellulaires. Il existe une forte similitude entre les macropinosomes et les "phagosomes spacieux" induits par des bactéries entéropathogènes telles que Salmonella et Shigella. Les macropinosomes de forment préférentiellement au front de migration et peuvent fusionner avec les lysosomes ou recycler leur contenu vers le milieu extracellulaire (régurgitation). La transformation de fibroblastes Rat-1 par les oncogènes v-Src ou K-Ras induit la formation constitutive de macropinosomes au niveau des zones du "ruffling". Notre travail s'est intéressé à la dynamique de cette macropinocytose constitutive et à ses régulateurs potentiels. La première partie de ma thèse concerne le sort des macropinosomes dans les fibroblastes transformés par v-Src. Ceux-ci ne fusionnent pas avec les endosomes contenant la transferrine. Pour tester l'effet de l'AMP-cyclique sur cette macropinocytose, nous avons utilisé deux analogues perméants de l'AMPc, le dibutyryl AMP-cylique et le 8-bromo-AMP-cyclique, ainsi qu'un activateur de l'adénylate cyclase, la forskoline. Ces trois agents ralentissent d'environ 35% la vitesse de l'accumulation nette d'un traceur de l'endocytose fluide, la peroxydase de raifort, dans les cellules transformées par v-Src, mais n'ont pas d'effet dans la lignée parentale. Cependant, au contraire d'inhibiteurs de la phospho-inositide 3-kinase (PI3K) ou d'inhibiteurs de la phospholipase C spécifique du phosphatidylinositol (PI-PLC), le dibutyryl AMP-cyclique ne ramène pas le taux d'accumulation de la peroxydase des fibroblastes transformés par v-Src au niveau des fibroblastes parentaux et n'empêche pas la formation de macropinosomes, visualisée en microscopie confocale. L'analyse détaillée de la cinétique d'accumulation de la peroxydase dans les cellules transformées a révélé que le dibutyryl AMP-cyclique ralentit l'accumulation de la peroxydase seulement après un temps supérieur à 5 minutes, en accélérant sa régurgitation, sans affecter le recyclage de la transferrine. Ces résultats montrent clairement que, dans les fibroblastes transformés par v-Src, la macropinocytose et la micropinocytose sont deux voies distinctes et que l'AMP-cyclique n'affecte ni la micropinocytose, ni la formation de macropinosomes, mais active sélectivement la régurgitation à partir de ceux-ci. La seconde partie de ma thèse s'intéresse à la régulation de la formation des macropinosomes. La transformation des fibroblastes Rat-1 par les oncogènes v-Src ou K-Ras ainsi que la transfection stable par un vecteur d'expression de la sous-unité régulatrice sauvage p85a, créant un ²dominant-positif² de la PI3K, provoquent la destruction des câbles de tension, le recrutement de l'actine corticale et la formation du ²ruffling² qui génère les macropinosomes, ce qui est reflété par l'accélération sélective de l'endocytose en phase fluide. Ces altérations corrèlent étroitement avec l'activation de la PI3K et de la PI-PLC, mesurées par la production des phospho-inositides D3 in situ et in vitro et par le niveau de l'IP3 respectivement, et sont abolies après transfection stable par un vecteur d'expression pour la sous-unité p85a tronquée, agissant comme ²dominant-négatif² de la PI3K, ou après traitement des cellules avec les inhibiteurs pharmacologiques de la PI3K et de la PI-PLC. Ceci démontre que ces deux enzymes sont nécessaires dans la signalisation de la macropinocytose constitutive. Ces inhibiteurs diminuent fortement le pourcentage des cellules montrant un ²ruffling² membranaire mais n'affectent pas l'endocytose de la transferrine. La réorganisation du cytosquelette d'actine dans les cellules transformées par v-Src ou dans le ²dominant-positif² de la PI3K est reversée après 30 à 60 minutes de traitement par un inhibiteur de la PI3K, la wortmannine. L'activité de la PI3K n'est pas modifiée par le traitement des cellules avec un inhibiteur de la PI-PLC. En revanche, l'activité de cette dernière est abolie par les inhibiteurs de la PI3K et chez le ²dominant-négatif² de la PI3K. Ceci permet de placer la PI-PLC en aval de PI3K dans la même voie de signalisation de la macropinocytose. En conclusion, une activation permanente et séquentielle de la PI3K et de la PI-PLC est nécessaire au remodelage du cytosquelette d'actine qui produit le ²ruffling² membranaire et la macropinocytose constitutive dans les fibroblastes transformés.

This study focuses on the mechanism of transmembrane signaling by Tar, the aspartate chemoreceptor of Escherichia coli. Like other bacterial chemoreceptors, Tar localizes to the cell membrane and relays information about the external chemical environment through the membrane to a cytoplasmic signaling domain. The output of the signaling domain controls the directional bias of the rotary flagellar motors of the cell. Net movement of a cell in a chemical gradient involves temporal comparison of the current concentration with the concentration in the recent (a few seconds) past. The current concentration is measured as the percent occupancy of the extracellular ligand-binding domain of the receptor, and the past is represented by the extent of covalent methylation of four conserved glutamyl residues in the cytoplasmic domain. Under steady-state conditions, the methylation level corresponds to ligand occupancy. Tar is a dimer, and much evidence suggests that dimers associate into trimers of dimers. Higher-order arrays of receptors form in the presence of the cytoplasmic proteins CheA and CheW. The conformational change generated by ligand binding is transmitted through the membrane by one subunit of a dimer. To examine whether this initially asymmetric signal becomes symmetric within the cytoplasmic domain, I examined aspartate-induced adaptive methylation of the two subunits of mutant Tar receptor heterodimers. In the presence of CheA and CheW, adaptive methylation after addition of aspartate was symmetric, but in their absence, although the level of methylation increased, the rates were different for the two subunits. I also found that cross-talk, at the level of adaptive methylation, occurs between different receptor types even in the absence of CheA and CheW. These results provide support for the idea that a tight association of receptor dimers within trimers of dimers allows for an actively signaling receptor to affect the methylation state, and thus presumably the signaling state, of receptors within a trimer that are not bound to an attractant ligand.

Summary : Macropinocytosis refers to the formation of large primary endocytic vesicles of irregular size and shape, generated by actin-driven evaginations of the plasma membrane, whereby cells avidly incorporate extracellular solutes. Macropinosomes resemble "empty" phagosomes and show no difference with the "spacious phagosomes" triggered by the enteropathogenic bacteria Salmonella and Shigella. Macropinosomes are formed at the leading edge and appear tightly regulated. They may fuse with lysosomes or regurgitate their content back to the extracellular space. Transformation of fibroblasts by Src or Ras results into the constitutive formation of macropinosomes at "ruffling" zones. My thesis adresses the dynamic of the constitutive macropinocytosis and its potential regulators. The first part of the thesis deals with the fate of macropinosomes. We found that, in v-Src transformed fibroblasts, macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase, forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer, horseradish peroxidase, in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phosphatidylinositol 3-kinase (PI3K) inhibitors and phosphatidylinositol-specific phospholipase C (PI-PLC), dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of peroxidase entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited its accumulation only after intervals >5 minutes, due to accelerated regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom. The second part of my thesis deals with the regulation of macropinocytosis. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection with an expression vector for wild-type PI3K regulatory subunit p85a, acting as dominant-positive PI3K, constitutively lead to stress fibers disruption, cortical actin recruitment, extensive ruffling and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and PI-PLC, as assayed by 3-phosphoinositides synthesis in situ and in vitro and IP3 steady state levels, respectively, and were abolished by stable transfection of Src-transformed cells with an expression vector for truncated p85a, acting as dominant-negative PI3K, as well as by pharmacological inhibitors of PI3K and PI-PLC, indicating requirement for both enzymes. These inhibitors strongly decreased the population of cells showing active membrane ruffling and did not detectably affect receptor-mediated endocytosis of transferrin. Interestingly, the effect of Src and dominant-positive p85a transfection on the actin cytoskeleton could be reversed within 30-60 min by the pharmacological inhibitor of PI3K, wortmannin. Whereas PI3K activation resisted PI-PLC inhibition by NCDC, PI-PLC activation was abolished by the PI3K inhibitor wortmannin and upon dominant-negative PI3K transfection, thus placing PI-PLC downstream of PI3K. Taken together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of actin cytoskeleton in oncogene-transformed fibroblasts resulting into constitutive ruffling and macropinocytosis.Résumé : La macropinocytose désigne la formation de grandes vésicules endocytaires primaires de taille et de forme irrégulière, produites par évagination de la membrane péricellulaire, qui permet l'accumulation accélérée de solutés extracellulaires. Il existe une forte similitude entre les macropinosomes et les "phagosomes spacieux" induits par des bactéries entéropathogènes telles que Salmonella et Shigella. Les macropinosomes de forment préférentiellement au front de migration et peuvent fusionner avec les lysosomes ou recycler leur contenu vers le milieu extracellulaire (régurgitation). La transformation de fibroblastes Rat-1 par les oncogènes v-Src ou K-Ras induit la formation constitutive de macropinosomes au niveau des zones du "ruffling". Notre travail s'est intéressé à la dynamique de cette macropinocytose constitutive et à ses régulateurs potentiels. La première partie de ma thèse concerne le sort des macropinosomes dans les fibroblastes transformés par v-Src. Ceux-ci ne fusionnent pas avec les endosomes contenant la transferrine. Pour tester l'effet de l'AMP-cyclique sur cette macropinocytose, nous avons utilisé deux analogues perméants de l'AMPc, le dibutyryl AMP-cylique et le 8-bromo-AMP-cyclique, ainsi qu'un activateur de l'adénylate cyclase, la forskoline. Ces trois agents ralentissent d'environ 35% la vitesse de l'accumulation nette d'un traceur de l'endocytose fluide, la peroxydase de raifort, dans les cellules transformées par v-Src, mais n'ont pas d'effet dans la lignée parentale. Cependant, au contraire d'inhibiteurs de la phospho-inositide 3-kinase (PI3K) ou d'inhibiteurs de la phospholipase C spécifique du phosphatidylinositol (PI-PLC), le dibutyryl AMP-cyclique ne ramène pas le taux d'accumulation de la peroxydase des fibroblastes transformés par v-Src au niveau des fibroblastes parentaux et n'empêche pas la formation de macropinosomes, visualisée en microscopie confocale. L'analyse détaillée de la cinétique d'accumulation de la peroxydase dans les cellules transformées a révélé que le dibutyryl AMP-cyclique ralentit l'accumulation de la peroxydase seulement après un temps supérieur à 5 minutes, en accélérant sa régurgitation, sans affecter le recyclage de la transferrine. Ces résultats montrent clairement que, dans les fibroblastes transformés par v-Src, la macropinocytose et la micropinocytose sont deux voies distinctes et que l'AMP-cyclique n'affecte ni la micropinocytose, ni la formation de macropinosomes, mais active sélectivement la régurgitation à partir de ceux-ci. La seconde partie de ma thèse s'intéresse à la régulation de la formation des macropinosomes. La transformation des fibroblastes Rat-1 par les oncogènes v-Src ou K-Ras ainsi que la transfection stable par un vecteur d'expression de la sous-unité régulatrice sauvage p85a, créant un ²dominant-positif² de la PI3K, provoquent la destruction des câbles de tension, le recrutement de l'actine corticale et la formation du ²ruffling² qui génère les macropinosomes, ce qui est reflété par l'accélération sélective de l'endocytose en phase fluide. Ces altérations corrèlent étroitement avec l'activation de la PI3K et de la PI-PLC, mesurées par la production des phospho-inositides D3 in situ et in vitro et par le niveau de l'IP3 respectivement, et sont abolies après transfection stable par un vecteur d'expression pour la sous-unité p85a tronquée, agissant comme ²dominant-négatif² de la PI3K, ou après traitement des cellules avec les inhibiteurs pharmacologiques de la PI3K et de la PI-PLC. Ceci démontre que ces deux enzymes sont nécessaires dans la signalisation de la macropinocytose constitutive. Ces inhibiteurs diminuent fortement le pourcentage des cellules montrant un ²ruffling² membranaire mais n'affectent pas l'endocytose de la transferrine. La réorganisation du cytosquelette d'actine dans les cellules transformées par v-Src ou dans le ²dominant-positif² de la PI3K est reversée après 30 à 60 minutes de traitement par un inhibiteur de la PI3K, la wortmannine. L'activité de la PI3K n'est pas modifiée par le traitement des cellules avec un inhibiteur de la PI-PLC. En revanche, l'activité de cette dernière est abolie par les inhibiteurs de la PI3K et chez le ²dominant-négatif² de la PI3K. Ceci permet de placer la PI-PLC en aval de PI3K dans la même voie de signalisation de la macropinocytose. En conclusion, une activation permanente et séquentielle de la PI3K et de la PI-PLC est nécessaire au remodelage du cytosquelette d'actine qui produit le ²ruffling² membranaire et la macropinocytose constitutive dans les fibroblastes transformés.

SH2-containing inositol polyphosphate 5-phosphatases, SHIP2, has been established as a regulator of the insulin cascade, of cell adhesion and spreading, actin structures, remodelling and cytoskeletal organization. However, the molecular mechanisms underlying these processes still needed additional investigations. Among different regulatory mechanisms, protein-protein interaction play an essential role. To better understand the molecular mechanism of SHIP2 in signalling pathway as well as to reveal novel roles of SHIP2, a two-hybrid was performed to search for SHIP2 protein interactors. JNK-interacting protein 1 (JIP1) and intersectin 1 (ITSN1) were two of the newly identified protein partners of SHIP2. In this thesis, we characterized the associations of SHIP2 with JIP1 and ITSN1 in different aspects as identifying the interacting domain involved, biochemical function regulations and cellular biological roles. The JIP scaffold family of proteins associate with MAPK, MAPKK and MAPKKK creating functional signaling modules to control the specificity of signal transduction. JIP1 is characterized as a scaffold protein assembling JNK, MAPK kinase 7 (MKK7), mixed lineage kinase (MLK), dual leucine zipper-bearing kinase (DLK). It thus enhances the selectivity and effectiveness of kinase activation during JNK signaling. In this thesis, the SHIP2-JIP1 interaction has been confirmed both in overexpression system in COS-7 and CHO-IR cells, and in native cells of COS-7. Both the proline-rich (PR) domain (residues 359-487) and PTB domain of JIP1 participated in this interaction. Overexpression of SHIP2 in COS-7 cells up-regulated JIP1-mediated JNK activation and the tyrosine phosphorylations of both JIP1 and MLK3. These effects were independent of SHIP2 catalytic activity. By the use of kinase inhibitors, we showed that Abl and Src family tyrosine kinases might be implicated in the regulation of JIP1 tyrosine phosphorylation. The residue Y270 of JIP1, a potential target of Abl tyrosine kinase, was shown to be involved in SHIP2-increased JIP1 tyrosine phosphorylation. In an in vitro assay, JIP1 negatively regulated the catalytic activity of SHIP2. In addition, upon the stimulation of okadaic acid, the overexpression of SHIP2 caused less viability of COS-7 cells. These data provide a new molecular link between SHIP2 and JIP1-mediated JNK pathway, and may help explain the biochemical mechanisms of SHIP2 in cellular apoptosis, as well as in insulin pathway. Another protein partner, ITSN1, is a multi-domain protein which plays a role in endocytosis, MAPK signalling and actin cytoskeleton. The interaction between SHIP2 and ITSN1 was confirmed in overexpression systems in COS-7 cells, as well as at the physiological concentration with the endogenously expressed proteins in C2C12 and COS-7 cells. EGF stimulation did not modulate the association of SHIP2 and ITSN1. ITSN1-SH3D, A, C and E domains interacted with the C-terminal part of SHIP2 with the binding affinity as SH3D>SH3A>SH3C>SH3E. Upon the stimulation of EGF, the expression of SHIP2 may recruit ITSN1 short form (ITSN1-S) to cell membrane. The ITSN-mediated ERK1/2 and JNK activations in response to EGF were not modulated when SHIP2 or catalytic mutant of SHIP2 or TSHIP2 was overexpressed. The link between SHIP2 and ITSN may provide one of the molecular mechanisms used by SHIP2 to participate in receptor endocytosis regulation. In conclusion, our data of the associations of SHIP2 with JIP1 and ITSN1 provide evidence for potential novel biochemical mechanisms of SHIP2 to be implicated in JNK pathway as well as EGF receptor endocytosis. JIP1 and ITSN1, which are both implicated in the JNK pathway, may also have a link through the common protein partner SHIP2, giving rise to potential interesting study goal.

This study focuses on the mechanism of transmembrane signaling by Tar, the aspartate chemoreceptor of Escherichia coli. Like other bacterial chemoreceptors, Tar localizes to the cell membrane and relays information about the external chemical environment through the membrane to a cytoplasmic signaling domain. The output of the signaling domain controls the directional bias of the rotary flagellar motors of the cell. Net movement of a cell in a chemical gradient involves temporal comparison of the current concentration with the concentration in the recent (a few seconds) past. The current concentration is measured as the percent occupancy of the extracellular ligand-binding domain of the receptor, and the past is represented by the extent of covalent methylation of four conserved glutamyl residues in the cytoplasmic domain. Under steady-state conditions, the methylation level corresponds to ligand occupancy. Tar is a dimer, and much evidence suggests that dimers associate into trimers of dimers. Higher-order arrays of receptors form in the presence of the cytoplasmic proteins CheA and CheW. The conformational change generated by ligand binding is transmitted through the membrane by one subunit of a dimer. To examine whether this initially asymmetric signal becomes symmetric within the cytoplasmic domain, I examined aspartate-induced adaptive methylation of the two subunits of mutant Tar receptor heterodimers. In the presence of CheA and CheW, adaptive methylation after addition of aspartate was symmetric, but in their absence, although the level of methylation increased, the rates were different for the two subunits. I also found that cross-talk, at the level of adaptive methylation, occurs between different receptor types even in the absence of CheA and CheW. These results provide support for the idea that a tight association of receptor dimers within trimers of dimers allows for an actively signaling receptor to affect the methylation state, and thus presumably the signaling state, of receptors within a trimer that are not bound to an attractant ligand.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 85-112).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains x, 92 p.: ill. Includes abstract and vita. Advisor: Clay B. Marsh, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 84-92).