Green fluorescent protein (GFP) tagged to the cytoplasmic tail of αIIb or β3 allows the expression of a fully functional integrin αIIIbβ3:: effect of β3GFP on αIIbβ3 ligand binding

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alpha IIb or 3 allowed normal expression, heterodimerization, processing and surface exposure of alpha IIb(GFP)beta3 and aIIb beta3(GFP) receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alpha IIb beta3 capping revealed an intense autofluorescent cap corresponding to unlabelled immuno-clustered GFP-tagged aIIb beta3. GFP-tagged aIIb beta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(PAK) tyrosine phosphorylation similar to wild-type alpha IIb beta3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alpha IIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alpha IIb beta3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alpha IIb beta3 during the early stages of cell attachment and spreading, starting with alpha IIb beta3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alpha IIb beta3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alpha IIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the 3 integrin cytoplasmic tail, rather than the alpha IIb subunit, plays a major role in alpha IIb beta3 affinity modulation. With the successful direct visualization of functional aIIb beta3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.