We present a general strategy for the dominant negative reduction in the levels of type-1 membrane-bound heterodimeric proteins within the secretory pathway through fusion of the soluble ectodomain of one of the partners to the transmembrane-cytosolic tail of the lysosomal protein Lamp1. Thus, in human embryonic kidney (HEK)-293 cells, overexpression of an integrin β3Lamp1 chimera resulted in a drastic reduction of its endogenous partner, the integrin αv subunit. The mechanism involves the formation in the endoplasmic reticulum of a αv/β3Lamp1 complex that is subsequently sorted towards a lysosomal/endosomal degradation pathway. The specificity of this approach is afforded by the invariance in the levels of the endogenous integrins α5 and β1 as compared with control cells. Conversely overexpression of integrin β3 in HEK-293 cells led to an increased level of αvβ3 at the cell surface. Functionally β3Lamp1 and β3 overexpressors exhibit decreased and increased adhesion to vitronectin, respectively, as well as diminished cellular aggregation. The application of this technology should enable the analysis of the functional importance of homodimers or heterodimers in the cell types of choice and the identification of novel partner proteins by proteomic approaches.
Down-regulation of alphav/beta3 integrin via misrouting to lysosomes by overexpression of a beta3Lamp1 fusion protein
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Magali CONESA, Annik PRAT, John S. MORT, Jacques MARVALDI, Jean-Claude LISSITZKY, Nabil G. SEIDAH; Down-regulation of alphav/beta3 integrin via misrouting to lysosomes by overexpression of a beta3Lamp1 fusion protein. Biochem J 1 March 2003; 370 (2): 703–711. doi: https://doi.org/10.1042/bj20021301
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