FGF-2 facilitates binding of SH3 domain of PLC-gamma 1 to vinculin and SH2domains to FGF receptor in corneal endothelial cells

PURPOSE: To determine the cellular localization of the Src homology (SH)2 a nd SH3 domains of PLC-gamma 1 and their cytoplasmic binding partners, livin g corneal endothelial cells were microinjected with the fusion proteins con taining SH domains. METHODS: Fusion proteins were prepared from plasmid vectors, and the fusion proteins containing SH2-SH2 [(SH2)2], SH2-SH2-SH3 [(SH2)2-SH3] or SH3 were isolated using affinity chromatography. Following microinjection, immunolo calization was analyzed using confocal laser microscope. RESULTS: Microinjected SH domains were targeted to the subcellular location following stimulation with FGF-2: the SH3 domain appeared to be targeted t o cytoskeleton; the (SH2)2 domain showed a dual localization in cytoplasm a nd plasma membrane; the (SH2)2-SH3 domain was predominantly localized at me mbrane and perinuclear sites. In the absence of stimulation by FGF-2, the m icroinjected fusion proteins remained at the injection sites. When cytoplas mic binding partners were determined by double-staining, the SH3 domain dem onstrated colocalization with vinculin: the staining profile of the SH3 dom ain was identical to that of vinculin, which demonstrates characteristic pu nctated profiles. The punctated staining of SH3 disappears toward the basal membrane, while that of vinculin remains in all confocal optical sections. On the other hand, some fraction of the (SH2)2 domain was colocalized with FGF receptor at the membrane site. When PLC-gamma 1 and F-actin were doubl e-stained, the endogenous PLC-gamma 1 demonstrated a diffuse cytoplasmic st aining and/or perinuclear staining, while phalloidin staining demonstrated that all cells have filamentous cytoplasmic distribution of F-actin. CONCLUSIONS: These findings indicate that the SH3 domain directs PLC-gamma 1 to bind to vinculin and that the SH2 domains may mediate the binding of P LC-gamma 1 to receptor tyrosine kinase. Furthermore, they suggest that phos phorylation is not required for targeting of PLC-gamma 1 to membrane or cyt oskeleton sites.