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Title: Molecular and functional characterization of the immunoreceptors CD300d and CD300f / Caracterització molecular i funcional dels immunoreceptors CD300d I CD300f
Author: Comas Casellas, Emma
Director: Sayós Ortega, Joan
Espel Masferrer, Enric
Keywords: Nanomedicina
Issue Date: 8-Mar-2013
Publisher: Universitat de Barcelona
Abstract: [eng] The main goal of this thesis was to investigate the function of the endogenous CD300f. Initially, CD300f was described as an inhibitory receptor, although some data generated in our lab strongly suggested the possibility that the receptor could trigger activating signals in specific situations. It was planned to use two monoclonal antibodies (UPD1 and UPD2), that recognize the extracellular domain of CD300f in order to analyze the function of the endogenous CD300f. However, extracellular domain of CD300f has a high homology with the extracellular domain of CD300d, a new member of the CD300 family receptors. Thus, it was essential to check if UPD1 and/or UPD2 cross-reacted with CD300d. Unexpectedly, CD300d was unable to reach the cell surface in transfected cells, so it was not possible to check if the antibodies recognize it. That result prompted us to further investigate and characterize the CD300d receptor. Finally, the cloning and characterization of CD300d has contributed to complete the description of the human CD300 locus. Concretely, the CD300d gene spans a 12.5 kb region on chromosome 17 (position 17q25.1), has an open reading frame of 585 bp and encodes for a type I transmembrane protein of 194 amino acids. The molecule is composed by an immunoglobulin-like extracellular domain, a transmembrane domain containing a negative charged residue and a short cytoplasmic tail without known signaling motifs. CD300d has a predicted molecular mass of 21.5kDa, however presents two distinct mature forms of 30 and 34 KDa in transfected COS-7 cells. Both conformations are posttranslationally modified by N-glycosylations in the Ig domain. The CD300d receptor is expressed by primary monocytes and granulocytes. Conversely, T, B or NK lymphocytes do not express the receptor. Unpredictably, CD300d is retained in the endoplasmic reticulum of transfected cells. Even so, the interaction with the FcR-gamma adapter permits a slight surface recovery of the receptor in COS-7 cells but not in RBL-2H3 cells. In addition, CD300d is able to interact in vitro with the rest of known CD300 family receptors, with the exception of CD300c. Therefore, CD300d could play a role in the formation of CD300 complexes on the cell surface and consequently could modulate the state of activation of myeloid cells. The CD300d and CD300f immunoglobulin domains present a 71% of identity. Nevertheless, UPD1 and UPD2 mAb recognize exclusively the CD300f receptor. Surprisingly, the stimulation of the endogenous CD300f receptor induces the production of TNF pro-inflammatory cytokine in primary monocytes, giving support to the dual activity of the receptor, initially described as inhibitor. Furthermore, crosslinking of endogenous CD300f, in PMA differentiated U937 or THP-1 monocytic cells, induce the production of TNF-alpha and IL-1-beta pro-inflammatory cytokines. The activating functions of the CD300f could be mediated through its association with FcR-gamma and/or DAP12 adapter molecules, proved in vitro. The binding involves the transmembrane charged residue of the adapters and the transmembrane domain of the receptor. The expression of endogenous FcR-gamma nd DAP12 adapter molecules was upregulated, in U937 and THP-1 cells. In addition Syk kinase, JNK kinase and PKC-delta are involved in the activating signaling mediated by CD300f. Altogether, supports the functional association of FcR-gamma and/or DAP12 with the CD300f receptor. Finally, we prove the existence of soluble forms of the CD300f receptor.
[cat] L'objectiu principal d’aquesta tesis era investigar la funció del receptor CD300f endogen. Inicialment, el CD300f es va descriure com a receptor inhibidor, tot i que alguns resultants del nostre laboratori suggerien la possibilitat que el receptor podia desencadenar respostes cel•lulars activadores en algunes circumstàncies. Amb el propòsit d’estudiar la funció del receptor CD300f endògen, es va decidir usar dos anticossos monoclonals (UPD1 i UPD2), que tenien la capacitat de reconèixer el domini extracel•lular del CD300f. Ambdós anticossos es varen obtenir en el nostre laboratori anteriorment, amb l’objectiu de permetre caracteritzar el receptor CD300f; varen ser seleccionats segons la seva capacitat per reconèixer el CD300f i no cross-reaccionar amb la resta de molècules CD300 conegudes fins al moment (CD300a, CD300b, CD300c i CD300e). Tot i així, poc temps abans de començar a treballar en aquesta tesis, en el laboratori s’havia clonat un nou membre de la família CD300, anomenat CD300d. Així doncs era necessari comprovar si els anticossos UPD1 o UPD2 cross-reaccionaven amb aquest nou membre de la família. Malauradament, el receptor CD300d era incapaç d’expressar-se a la membrana de cèl•lules transfectades, així doncs no era possible verificar si els anticossos el reconeixien. Aquest resultat ens va portar a investigar en més profunditat el CD300d. Finalment, el clonatge i caracterització del receptor CD300d ha contribuït a la descripció del locus humà CD300, que conté sis membres (del CD300a al CD300f) i ocupa una regió de 450 Kb en el cromosoma 17. Es va poder confirmar que els anticossos UPD1 i UPD2 reconeixien específicament el CD300f i no el CD300d. Sorprenentment, es va demostrar que l’estimulació del receptor CD300f endògen en monocits primaris o les línies monocítiques U937 i THP-1 generaven una resposta pro-inflamatòria. En referència al receptor CD300d es va observar que no s’expresa a nivell de membrana cel•lular sinó que era retingut al reticle endoplasmàtic, indicant que la seva funció biològica podria ser la de regular l’expressió superficial de la resta de membres de la família CD300.
Note: Tesi realitzada a l'Institut de Recerca de l'Hospital Universitari Vall d'Hebron - Grup d'Immunologia / CIBBM-Nanomedicina
Appears in Collections:Tesis Doctorals - Facultat - Biologia

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