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Murine CD248 and its cytoplasmic domain : characterising a novel tumour and inflammation marker

University of British Columbia

CD248 is a member of a family of transmembrane glycoproteins containing an N-terminal C-type lectin-like domain. This family includes thrombomodulin and CD93, proteins known to modulate immunity, cell proliferation and homeostasis. CD248 is expressed in perivascular and stromal cells, during embryonic development and post-natally during inflammation and cancer. In the mesenchymal compartment of most normal adult tissues, CD248 expression is not detectable. It is believed that CD248 is involved in cross-talk between endothelial cells and pericytes, thereby playing a role in growth, metastasis and angiogenesis associated with the development of tumours and inflammatory lesions. Intracellular signaling, mediated via the cytoplasmic domain of CD248, promotes tumour growth and inflammation. CD248 knock-out mice and mice expressing CD248 lacking the cytoplasmic domain were resistant to tumour growth and inflammatory arthritis. Although signaling pathways have not been delineated, examination of the cytoplasmic domain of CD248 reveals three highly conserved putative phosphorylation sites and a PDZ-binding motif. We hypothesised that these structural features are important for CD248 function. We generated a variety of murine CD248 pcDNA constructs that encode CD248 with mutations in the cytoplasmic domain. We confirmed that all the mutants were transcribed and translated. Mutant proteins were expressed on the cell surface, in a similar manner to wildtype CD248. Introduction of some mutant forms CD248 into cells caused CD248 to exhibit different intracellular localisation and induced changes in cellular morphology compared to wildtype. Limited functional studies demonstrated CD248-dependent alterations in cellular MMP-9 production. The findings underline an important role of CD248’s cytoplasmic domain in regulating cellular morphology and function that may impact its role in health and disease. Extracellular interacting partners for CD248 have been previously described. Searches for intracellular partners interacting with the cytoplasmic domain have been less successful. Our results strongly suggest that these exist. Co-immunoprecipitation studies have revealed several putative interacting proteins that set the stage for future confirmatory and functional analyses. The cytoplasmic domain of CD248 is important to study as it holds much promise as a therapeutic target for proliferative disorders. The information gathered in this project may be used to delineate clinically relevant CD248 signaling pathways.

Text

2012-06-27

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/42573

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/