Binding and patching of HLE in response to the fusion domain of HIV. (A) Wells in a microtiter plate were uncoated (▪) or were coated (•) with 10 mM HIV-1 fusion peptide (FLGFL) and incubated with ¹²⁵I-U937 clone 10 solubilized cytoplasmic membrane proteins. Hypertonic saline (0.5 M NaCl) was used to limit nonspecific ionic interactions between solubilized membranes and the hydrophobic peptide. Maximum bound cpm/well at saturation was 1.3 × 10⁵ (r² = 0.94), Binding to uncoated wells was 9.0 ± 0.3 × 10³ cpm. Specific binding to fusion peptide is represented as the difference between coated and uncoated wells, and binding to uncoated wells is depicted for comparison. (B) Scatchard analysis revealed a linear character (r² = 0.81) to the interactions and association constant 1 × 10³ M^-1 in hypertonic saline. Interaction of cell-surface HLE with FLGFL (C) or α₁PI (D) stimulated patching. Bound monoclonal antibody specific for HLE was detected using biotinylated anti-IgG and FITC-conjugated streptavidin. Significantly, HLE (green) was found to copatch with CXCR4 (red) in response to crosslinked polyclonal (E) or monoclonal (F) anti-HLE. Cells were stimulated, fixed, stained, and examined by suspension under coverslip. Negative-control cells stained for HLE in the absence of HIV-1 fusion peptide, α₁PI, or other HLE ligands depicted no detectable staining (not shown). (G) Colocalization of α₁PI (green) and SDF-1α (red) on the attached surface of clone 10 was detected when cells were stimulated by α₁PI and postincubated with SDF-1α. (H) In contrast, when cells were first stimulated with SDF-1α and postincubated with α₁PI, SDF-1α was detected in an internal section, but α₁PI was not detected in any section. In the cell represented, SDF-1α was detected 2.4 μm from the attached surface of clone 10. Cells were stimulated, slides were prepared by cytospin, fixed, and stained. Colocalization by CLSM was analyzed using 8-step 0.6-μm sectional scanning from the attached surface toward the unattached surface of the cells. Patching and colocalization were performed at least 3 times and examined by 2 investigators independently. A representative cell is depicted. All photomicrographs were magnified to the same scale; bar represents 25 μm. Homogeneous receptor density at equilibrium was achieved by exposing cells to α₁PI by culturing in medium containing 10% FBS prior to staining.