Down-regulation of alpha v/beta 3 integrin via misrouting to lysosomes by overexpression of a beta 3Lamp1 fusion protein.
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 beta 3Lamp1 chimera resulted in a drastic reduction of its endogenous partner, the integrin alpha v subunit. The mechanism involves the formation in the endoplasmic reticulum of a alpha v/beta 3Lamp1 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 alpha 5 and beta1 as compared with control cells. Conversely overexpression of integrin beta 3 in HEK-293 cells led to an increased level of alpha v beta 3 at the cell surface. Functionally beta 3Lamp1 and beta 3 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.
Conesa
Magali
M
UMR CNRS 6032, Faculté de Pharmacie, 27 Bd J. Moulin, 13385 Marseille, Cedex 5, France.
Prat
Annik
A
Mort
John S
JS
Marvaldi
Jacques
J
Lissitzky
Jean-Claude
JC
Seidah
Nabil G
NG
D015703:Antigens, CD / Q000235:genetics*; D015536:Down-Regulation / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D039564:Integrin alphaV; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D039661:Integrin beta3 / Q000235:genetics*; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D015703:Antigens, CD; D039564:Integrin alphaV; D039302:Integrin alphaVbeta3; D039661:Integrin beta3; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D011993:Recombinant Fusion Proteins