FGF-2 facilitates binding of SH3 domain of PLC-gamma1 to vinculin and SH2 domains to FGF receptor in corneal endothelial cells.

PURPOSE

To determine the cellular localization of the Src homology (SH)2 and SH3 domains of PLC-gamma1 and their cytoplasmic binding partners, living corneal endothelial cells were microinjected with the fusion proteins containing 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, immunolocalization 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 to cytoskeleton; the (SH2)2 domain showed a dual localization in cytoplasm and plasma membrane; the (SH2)2-SH3 domain was predominantly localized at membrane and perinuclear sites. In the absence of stimulation by FGF-2, the microinjected fusion proteins remained at the injection sites. When cytoplasmic binding partners were determined by double-staining, the SH3 domain demonstrated colocalization with vinculin: the staining profile of the SH3 domain was identical to that of vinculin, which demonstrates characteristic punctated 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-gamma1 and F-actin were double-stained, the endogenous PLC-gamma1 demonstrated a diffuse cytoplasmic staining 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-gamma1 to bind to vinculin and that the SH2 domains may mediate the binding of PLC-gamma1 to receptor tyrosine kinase. Furthermore, they suggest that phosphorylation is not required for targeting of PLC-gamma1 to membrane or cytoskeleton sites.