| Two members of the immunoglobulin superfamily, FcgammaRIIb and Thy-1, have been studied using biophysical techniques. Membrane binding of the cytoplasmic tails of FcgammaRIIb1 and b2 and their phosphorylation by Lyn kinase have been determined. Membrane binding experiments were performed with purified, fluorescently-labeled cytoplasmic regions and various lipid compositions. After using extensive fluorescence techniques, including fluorescence emission, fluorescence anisotropy, fluorescence resonance energy transfer, and total internal reflection fluorescence microscopy, it was determined that the cytoplasmic tails of FcgammaRIIb do not bind lipid bilayers.; In vitro phosphorylation assays were developed to test whether membrane colocalization is required for the phosphorylation of FcgammaRIIb by Lyn kinase in vivo and to determine the phosphorylation pattern of FcgammaRIIb in vitro. Purified cytoplasmic regions of FcgammaRIIb1 and b2 were incubated with recombinant Lyn kinase and analyzed by SDS-PAGE and western blots. The cytoplasmic region of FcgammaRIIb1 was found to be phosphorylated on its ITIM tyrosine, while the cytoplasmic region of FcgammaRIIb2 was not phosphorylated. The SH3 domain of Lyn kinase binds a PxxPxxR motif which may be necessary for its activation. FcgammaRIIb1 has this sequence in its cytoplasmic region, while FcgammaRIIb2 does not. These data suggest that membrane colocalization is not required for the phosphorylation of FcgammaRIIb1 by Lyn kinase and the PxxPxxR motif in its cytoplasmic region facilitates its phosphorylation.; The density-dependent diffusion of a GPI-anchored protein, Thy-1, is reported. Thy-1 was purified, fluorescently labeled, and reconstituted into liposomes used to make substrate supported planar membranes. Fluorescence pattern photobleaching recovery was used to measure the lateral diffusion of Thy-1 in the membrane. The diffusion coefficient of Thy-1 decreased from 4.4 +/- 0.17 to 1.7 +/- 0.02 mum2/sec as the density increased from 1,100 to 56,000 molec/mum2. The fractional recovery remained nearly constant. These data will be used to test existing theories and models for protein diffusion in membranes. |
