Green fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.

摘要: Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in living cell. GFP fused frame to cytoplasmic tail either alphaIIb or allowed normal expression, heterodimerization, processing and surface exposure alphaIIbGFPbeta3 alphaIIb(beta3)GFP receptors Chinese hamster ovary (CHO) cells. Direct microscopic observation cells suspension following antibody-induced alphaIIb(beta3) capping revealed intense cap corresponding unlabelled immunoclustered GFP-tagged alphaIIb(beta3). alphaIIbbeta3 mediated fibrinogen-dependent cell adhesion, were readily detectable focal adhesions unstained triggered p125(FAK) tyrosine phosphorylation similar wild-type (where FAK corresponds adhesion kinase). However, tagged beta3, but not alphaIIb, induced spontaneous CHO aggregation presence soluble fibrinogen, well binding fibrinogen mimetic monoclonal antibody PAC1 absence receptor activation. Time-lapse imaging transfectants characteristic redistribution during early stages attachment spreading, starting with clustering at rim contact area, that gradually overlapped boundary attached cell, and, onset reorganization adhesions. Taken together, our results demonstrate (1) fusion subunits allows expression functional receptor, (2) structural modification tail, rather than subunit, plays major role affinity modulation. With direct cells, generation integrins transgenic animals will become possible, allowing new approaches study dynamics functions.