Expression and functional characterisation of the collagen receptor glycoprotein VI
This thesis is concerned with the study of the collagen receptor glycoprotein VI (GPVI). GPVI has been studied in a number of human haematopoietic cell lines and found to be expressed only in those showing mekaryocytic features. Moreover, differentiation of the human cell lines HEL and CMK to a megakaryocytic lineage using the phorbol ester PMA revealed upregulation of GPVI together with the associated FcR γ-chain. Further, primary cultures of mouse marrow cells demonstrated up-regulation of GPVI towards the end of differentiation, therefore confirming data obtained with cell lines and pointing to GPVI as a possible marker of megakaryocytic differentiation. Structure/function studies of GPVI were carried out by means of transient and stable transfection of the receptor into COS-7 or K562 cells. These studies demonstrated the necessity of the transmembrane argine and the cytoplasmic tail of GPVI for association to the FcR γ-chain. Lack of association or absence of FcR γ-chain rendered GPVI unable to signal, despite binding to convulxin, a GPVI-specific ligand which causes activation of the receptor. K562 cells expressing GPVI and the FcR γ-chain were able to reconstitute the proximal but not distal events in GPVI signaling. A detailed analysis demonstrated impairment in phosphorylation and translocation of SLP-76 to the membrane, despite the presence and activation of others proteins known to be necessary for this phosphorylation/translocation to occur. Stable expression of GPVI in K562 cells, which display low levels of expression of the collagen receptors α2βl and CD36, does not confer signaling properties in response to collagen. However, the cells respond to a collagen related peptide (CRP) which is specific for GPVI and to the snake venoms convulxin, alborhagin and alboaggregin-A, demonstrating that GPVI is one important component through which these venoms are acting.