Localization of plasma membrane proteins in lipid rafts during signaling

Abstract

We have examined the preferential trafficking of membrane proteins to specialized, cholesterol-enriched microdomains within the plasma membrane termed lipid rafts. Isopycnic sucrose gradient ultracentrifugation was used to subfractionate membrane fragments. Membrane proteins of interest within low or high buoyant density membrane fractions were identified on Western blots prepared from sucrose fractions. To independently confirm these results, we used other methods including single-particle tracking to characterize the confinement of individual membrane proteins within small membrane compartments. In studies of the luteinizing hormone receptor (LHR), a receptor involved in male and female reproductive function, we demonstrated that the LHR moves into cholesterol-enriched lipid rafts upon binding of its physiological ligand, human chorionic gonadotropin (hCG). Only LHRs capable of receptor-receptor interactions translocate into these membrane structures. In studies of membrane proteins involved in allergen-mediated signaling in mast cells and basophils, we showed that the mast cell function-associated antigen (MAFA) is constitutively associated with the type I Fϲε receptor (FϲεRI) and that, upon crosslinking of either MAFA or FϲεRI, these membrane proteins translocate together into rafts. Together these studies suggest that rafts may be physiologically-relevant membrane structures involved in cell signaling events mediated by these membrane proteins. The role of protein aggregation within the plasma membrane in ligand-mediated signaling and translocation of membrane proteins into rafts is also discussed.