Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

The CGRP1 receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to Gs. R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an α helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. 1290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function. © 2006 American Chemical Society.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

Direction of outward FDI of EMNEs:evidence from the Indian pharmaceutical sector

This article seeks to add to the small but growing literature of emerging-market multinational enterprises (EMNEs). Using two linked large firm-level databases, it seeks to explore the determinants of outward investment of Indian pharmaceutical companies, distinguishing between developed- versus developing-country destinations. It specifically examines the impact of two firm-level characteristics that embody “non-OLI” [ownership, location, and internalization] firm-specific capabilities of EMNEs. The finding of this study is that family firms are keen on investing in other developing countries but much less so in developed countries. However, international linkages in the form of foreign investors offset this.

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Making sense of the ‘difficult’ past:transmission of political heritage and memory-work among young people across Europe

This article considers young people’s socialization into mnemonic communities in 14 European countries. It argues that such socialization is an intersubjective and selective process that, to a great degree, depends on the particular social environment that conditions the discourses on pasts available to young people. Drawing on memory studies, it recognizes memory as a valid alternative to the institutionalized past (history) but envisages the two as inextricably connected. Given this, it identifies several strategies adopted by young people in order to socialize understandings of the past. While these strategies vary, some reveal receptivity to populist and far right ideologies. Our study demonstrates how internalization of political heritage via mnemonic socialization within families is conditioned by both the national political agenda and socio-economic situation experienced across Europe.

Does the MK2-dependent production of TNFα regulate mGluR-dependent synaptic plasticity?

The molecular mechanisms and signalling cascades that trigger the induction of group I metabotropic glutamate receptor (GI-mGluR)-dependent long-term depression (LTD) have been the subject of intensive investigation for nearly two decades. The generation of genetically modified animals has played a crucial role in elucidating the involvement of key molecules regulating the induction and maintenance of mGluR-LTD. In this review we will discuss the requirement of the newly discovered MAPKAPK-2 (MK2) and MAPKAPK-3 (MK3) signalling cascade in regulating GI-mGluR-LTD. Recently, it has been shown that the absence of MK2 impaired the induction of GI-mGluR-dependent LTD, an effect that is caused by reduced internalization of AMPA receptors (AMPAR). As the MK2 cascade directly regulates tumour necrosis factor alpha (TNFα) production, this review will examine the evidence that the release of TNFα acts to regulate glutamate receptor expression and therefore may play a functional role in the impairment of GI-mGluRdependent LTD and the cognitive deficits observed in MK2/3 double knockout animals. The strong links of increased TNFα production in both aging and neurodegenerative disease could implicate the action of MK2 in these processes.

Fejezetek a nemzetközi üzleti gazdaságtanból 4. = Chapters in International Business. Reviews on Selected Papers on International Business 4.

A kötet azzal a céllal készült, hogy a Nemzetközi vállalatgazdaságtant tanulmányozók számára további támpontokat és ismeretbővítési lehetőségeket adjon magyar nyelven. A műhelytanulmány a nemzetközi üzleti gazdaságtan (international business) öt témaköréről ad áttekintést: az internalizációs elmélet, az intézményi megközelítés, a külpiacra lépés kisvállalati nézőpontból, a leányvállalatok lehetséges szerepei, és végül módszertani megfontolások. A korábbi kötetekhez hasonlóan, a műhelytanulmány fejezeteit a BCE Gazdálkodástani Doktori Iskola PhD hallgatói készítették. ----- The aim of the working paper is to provide insights on selected areas in international business. It is a supplement for the Hungarian language IB textbook. Five fields are covered in the working paper: internalization theory and its extensions, forming institutions in host countries, challenges of foreign market entry for small businesses, roles of subsidiaries in the multinational corporations, and methodological issues in IB. The chapters in the working paper were prepared by the PhD students of the Business and Management PhD School at Corvinus University of Budapest.

„Mennyit ér” a fenntarthatóság? – Esettanulmány az externális hatások mérésére azok internalizálására (What is sustainability worth? – A case study of measuring and internalizing negative environmental effects of logistics operation)

A fenntarthatóság központi kérdés a gazdálkodástudományban, ezen belül a logisztikában, hiszen a környezetre rótt szennyezésben e vállalati működési területnek is jelentős a szerepe. A szerzők dolgozata egy konkrét hazai esettanulmány segítségével vizsgálja a kérdéskört. Azt elemzik, vajon egy, az Európai Unióban is új kamiontípus (az ún. EuroCombi) alkalmazása a logisztikai folyamatokban milyen externális hatásokkal jár. Elemzésükben kísérletet tesznek ezeknek az externális hatásoknak a pénzben kifejezett mérésére. Azért fontos mindez, mert e mérés és számszerűsítés nélkül lehetetlen ezeknek a hatásoknak az internalizálása, vagyis belsővé tétele. Így mérés nélkül sem az érintett vállalatok tulajdonosai, sem azok belső döntéshozói, de a külső szabályozó szereplők sem tudják bevonni e hatásokat gazdasági döntéseikbe. Ez a mérés nem egyszerű feladat, de nem is lehetetlen. Mindenképpen alapját, előfeltételét képezi azonban annak, hogy ilyen jellegű döntéseknél az érintettek a társadalmi, környezeti érdekeket is érvényesíteni tudják. ____ Sustainability is a key issue today both for practitioners and researchers. This is true especially for logisticians. Logistics is a function that has a significant CO2 emission and also destructs transportation infrastructure that burden our environment. The article is focusing on these issues with the help of a Hungarian case study. It analysis the effect of applying a new, long and heavy type of truck, called EuroCombi. It presents a methodology usable for measuring these negative external effects in Forint. Without proper measurement internalization of costs these externalities generate is impossible. As it will be presented in details, such a measurement is although not very easy but achievable.

Speaking Up: A Phenomenological Study of Student Perceptions of Being Silenced in their Higher Education Classrooms

This phenomenological study explored students’ experiences of being silenced in their higher education classroom. Themes emerging from this study include the influence of past experiences, devaluation of students’ previous knowledge and learning, use of communication patterns to silence students, and internalization of conflict and oppression resulting from being silenced.

Selective and specific activation of Rab5 during endocytosis of receptor tyrosine kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

The performance of high -ability Black upper elementary grade students in an International Baccalaureate program

The purpose of this study was to investigate the common factors and experiences that contribute to the success of high ability Black students enrolled in Frank C. Martin Elementary School, the first school in Florida authorized to offer the International Baccalaureate (IB) Primary Years Programme (PYP). The study further sought to determine ways in which educators and stakeholders assisted in maintaining and in increasing Black students' achievement that motivated and encouraged them to pursue similar programs at the middle and high school levels. ^ Three sources of data were used: (a) individual interviews with fourth and fifth grade high ability Black students using a semi-structured format elicited discussion of their perceptions of the PYP and factors contributing to their success; (b) individual interviews with their fourth and fifth grade teachers elicited discussion of teacher expectations and effective instructional strategies; and (c) a questionnaire asked parents of the participating students their reasons for choosing the PYP, their perceptions of the program, and their own level of involvement in their child's learning. Three separate focus groups gathered further data. ^ The results revealed that the factors contributing to the success of high ability Black elementary school students are consistent with those of students in other racial groups. These are a challenging program, high teacher and parental expectations, strong parental involvement and support, a celebration of culture and diversity in a caring and nurturing environment, and the development and internalization of positive attitudes. ^ Implications for future studies might include a longitudinal study conducted over seven years to trace the achievements of Black students throughout the entire IB Program. ^

On the developmental continuity /discontinuity of early adolescent patterns of racial identity salience: Relations with later identities and psychosocial adjustment

Given the significant amount of attention placed upon race within our society, racial identity long has been nominated as a meaningful influence upon human development (Cross, 1971; Sellers et al., 1998). Scholars investigating aspects of racial identity have largely pursued one of two lines of research: (a) describing factors and processes that contribute to the development of racial identities, or (b) empirically documenting associations between particular racial identities and key adjustment outcomes. However, few studies have integrated these two approaches to simultaneously evaluate developmental and related adjustment aspects of racial identity among minority youth. Consequently, relations between early racial identity developmental processes and correlated adjustment outcomes remain ambiguous. Even less is known regarding the direction and function of these relationships during adolescence. To address this gap, the present study examined key multivariate associations between (a) distinct profiles of racial identity salience and (b) adjustment outcomes within a community sample of African-American youth. Specifically, a person-centered analytic approach (i.e., cluster analysis) was employed to conduct a secondary analysis of two archived databases containing longitudinal data measuring levels of racial identity salience and indices of psychosocial adjustment among youth at four different measurement occasions.^ Four separate groups of analyses were conducted to investigate (a) the existence of within-group differences in levels of racial identity salience, (b) shifts among distinct racial identity types between contiguous times of measurement, (c) adjustment correlates of racial identity types at each time of measurement, and (d) predictive relations between racial identity clusters and adjustment outcomes, respectively. Results indicated significant heterogeneity in patterns of racial identity salience among these African-American youth as well as significant discontinuity in the patterns of shifts among identity profiles between contiguous measurement occasions. In addition, within developmental stages, levels of racial identity salience were associated with several adjustment outcomes, suggesting the protective value of high levels of endorsement or internalization of racial identity among the sampled youth. Collectively, these results illustrated the significance of racial identity salience as a meaningful developmental construct in the lives of African-American adolescents, the implications of which are discussed for racial identity and practice-related research literatures. ^

Regional diversification and performance: Conceptualization and competing strategic objectives

This dissertation comprised of three essays provides justification for the need to pursue research on multinationality and performance with a more fine-grained approach. Essay one is a conceptual response to an article written by Jean-Francois Hennart in 2011 which questions the need and approach toward future research in this domain. I argue that internalization theory does not render multinationality and performance research meaningless and identify key areas where methodological enhancements can be made to strengthen our research findings with regard to Hennart's call for more content validity. Essay two responds to the need for more-fine grained research on the consequences of multinationality by introducing non-traditional measures of performance such as social and environmental performance and adopting a more theoretically relevant construct of regionalization to capture international diversification levels of the firm. Using data from the world's largest 600 firms (based on sales) derived from Bloomberg and the Directory of Corporate Affiliates; I employ general estimating equation analysis to account for the auto-correlated nature of the panel data alongside multivariate regression techniques. Results indicate that regionalization has a positive relationship with economic performance while it has a negative relationship with environmental and social performance outcomes, often referred to as the "Triple Bottom-Line" performance. Essay three builds upon the work in the previous essays by linking the aforementioned performance variables and sample to corporate reputation which has been shown to be a beneficial strategic asset. Using Structural Equation Modeling I explore economic, environmental and social signals as mediators on relationship between regionalization and firm reputation. Results indicate that these variables partially mediate a positive relationship between regionalization and firm reputation. While regionalization positively affects the reputation building signal of economic performance, it aids in reputation building by reducing environmental and social disclosure effects which interestingly impact reputation negatively. In conclusion, the dissertation submits opportunities for future research and contributes to research by demonstrating that regionalization affects performance, but the effect varies in accordance with the performance criterion and context. In some cases, regional diversification may produce competing or conflicting outcomes among the potential strategic objectives of the firm.

Novel insights into the mechanisms of regulation of tyrosine kinase receptors by Ras Interference 1

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBP&agr;. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.