LOX-1 scavenger receptor mediates calcium-dependent recognition of phosphatidylserine and apoptotic cells.

The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) scavenger receptor regulates vascular responses to oxidized-low-density-lipoprotein particles implicated in atherosclerotic plaque formation. LOX-1 is closely related to C-type lectins, but the mechanism of ligand recognition is not known. Here we show that human LOX-1 recognizes a key cellular phospholipid, PS (phosphatidylserine), in a Ca2+-dependent manner, both in vitro and in cultured cells. A recombinant, folded and glycosylated LOX-1 molecule binds PS, but not other phospholipids. LOX-1 recognition of PS was maximal in the presence of millimolar Ca2+ levels. Mg2+ was unable to substitute for Ca2+ in LOX-1 binding to PS, indicating a Ca2+-specific requirement for bivalent cations. LOX-1-mediated recognition of PS-containing apoptotic bodies was dependent on Ca2+ and was decreased to background levels by bivalent-cation chelation, LOX-1-blocking antibodies or PS-containing liposomes. The LOX-1 membrane protein is thus a Ca2+-dependent phospholipid receptor, revealing novel recognition of phospholipids by mammalian lectins.

The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts.

Pleckstrin homology (PH) domains act to target proteins to the plasma membrane and intracellular vesicles by binding to specific phosphoinositol phospholipids. We have investigated the binding kinetics of PH domains found in the tail region of the molecular motor, myosin X. Using total internal reflection fluorescence microscopy, we observed binding and release of individual PH domains fused to green fluorescent protein at the plasma membrane of living cells. Individual spots of light corresponding to single fluorescently tagged molecules were imaged onto a sensitive camera system, and digital image processing was then used to identify each fluorophore and store its trajectory in time and space. The PH domains bound with an apparent on-rate of 0.03 microm(-1) microm(-2) s(-1) and a detachment rate constant of 0.05 s(-1). The average residency time of the domains at the plasma membrane was about 20s. We found very limited movement of the membrane-bound PH domains in the mouse myoblast cells that we studied. This implies that the PH domains must either be attached to the cytoskeleton or corralled in a lipid compartment. Localization of the PH domains together with their rapid detachment rate is probably important in controlling the response of myosin X to signaling events and in regulating its cellular function.