Clustering of Raft-Associated Proteins in the External Membrane Leaflet Modulates Internal Leaflet H-Ras Diffusion and Signaling

Abstract

One of the least-explored aspects of cholesterol-enriched domains (rafts) in cells is the coupling between such domains in the external and internal monolayers and its potential to modulate transbilayer signal transduction. Here, we employed fluorescence recovery after photobleaching to study the effects of antibodymediated patching of influenza hemagglutinin (HA) proteins [raft-resident wild-type HA and glycosylphosphatidylinositol-anchored HA, or the nonraft mutant HA(2A520)] on the lateral diffusion of internal-leaflet raft and nonraft Ras isoforms (H-Ras and K-Ras, respectively). Our studies demonstrate that the clustering of outer-leaflet or transmembrane raft-associated HA proteins (but not their nonraft mutants) retards the lateral diffusion of H-Ras (but not K-Ras), suggesting stabilized interactions of H-Ras with the clusters of raft-associated HA proteins. These modulations were paralleled by specific effects on the activity of H-Ras but not of the nonraft K-Ras. Thus, clustering raft-associated HA proteins facilitated the early step whereby H-Ras is converted to an activated, GTP-loaded state but inhibited the ensuing step of downstream signaling via the Mek/Erk pathway. We propose a model for the modulation of transbilayer signaling by clustering of raft proteins, where external clustering (antibody or ligand mediated) enhances the association of internal-leaflet proteins with the stabilized clusters, promoting either enhancement or inhibition of signaling.Cholesterol and sphingolipid-enriched domains termed "lipid rafts" were demonstrated in artificial lipid bilayers and proposed to exist in cell membranes (1,4,9,10,18,26,49,50). In spite of a wealth of studies on the functional importance of interactions with raft-like domains for many cellular processes (reviewed in references 17, 18, 26, 49, 50, and 53), many questions remain concerning the size, lifetime, and even the existence of lipid rafts in live cells (1,8,10,17,18,23,26,27,33,45,49,51,54). The original simplistic view of lipid rafts was as preexisting liquid-ordered domains enriched in cholesterol and sphingolipids, into which specific proteins partition preferentially (4, 10, 49). More recent views envision rafts as small and transient cholesterol-dependent assemblies complexed with proteins, where interactions among the specific proteins influence the formation, size, and stability of these structures (1,17,44). The differences between these views involve mainly the nature of cholesterol-dependent assemblies in resting cell membranes. The studies presented here focus not on the resting state but rather on effects triggered by the clustering of proteins associated with cholesterol-sensitive assemblies; therefore, we will refer to such assemblies throughout as "rafts," without implications to a specific model.Numerous reports provide evidence in support of such domains in both the outer and inner plasma membrane leaflets (7,18,26,44,50,53,55). The coupling between raft-like domains in different membrane leaflets has an intriguing potential...

Formation of Stable Homomeric and Transient Heteromeric Bone Morphogenetic Protein (BMP) Receptor Complexes Regulates Smad Protein Signaling

“…3). This is typical of transient interactions because FabЈ-labeled receptors can dissociate and reassociate with their immobilized counterparts several times during the FRAP measurement (41,42). The formation of heteromeric BMP receptor complexes, which appear here to be in dynamic exchange with other cell surface receptor populations, is in line with earlier immunofluorescence copatching and co-immunoprecipitation studies (18,25), which showed the existence of PFCs and ligand-mediated heteromeric complexes among type I and type II BMP receptors.…”

“…The lateral diffusion coefficient (D) and the mobile fraction (R f ) were extracted from the FRAP curves by nonlinear regression analysis, fitting to a lateral diffusion process (40). Patch/FRAP studies were performed similarly except that antibody-mediated cross-linking-patching of an HA-tagged BMP receptor (described above) preceded the measurement (41,42). This enables determination of the effects of immobilizing one receptor type on the lateral diffusion of the co-expressed receptor, allowing identification of complex formation between them and distinction between transient and stable interactions (41,42).…”

“…If the complex lifetimes are longer than the characteristic FRAP times, the IgG-mediated immobilization of one receptor type is expected to reduce R f of the uncross-linked receptor because bleached molecules of the latter would not undergo appreciable dissociation from the immobile patches during the FRAP measurement. On the other hand, if the complex lifetime is short relative to the FRAP time, each molecule of the FabЈ-labeled receptor would undergo several associationdissociation cycles during the recovery phase of the FRAP experiment, leading to a reduction in D rather than in R f (41,42). When the effect is on R f , the percentage of oligomerization among different receptors can be calculated from the percent reduction in R f of the uncross-linked receptor; for example, a reduction of R f from 0.80 to 0.60 upon patching of the co-expressed receptor implies 100 ϫ (0.20/0.80) ϭ 25% oligomerization.…”

“…A limitation of the method is that it yields information exclusively on the mobile receptor population; thus, no information can be derived on the receptor population that is immobile already prior to the patching step. It should be noted that when the effect is on D the transient nature of the interactions precludes an exact calculation of the percentage of oligomerization because the extent of retardation in the apparent D value depends not only on the concentrations of complexed versus free receptors but also on the binding-unbinding rates of the FabЈ-tagged receptor to the co-expressed immobilized receptors relative to the lateral diffusion rate (41,42).…”

“…However, both methods can only detect relatively stable complexes, whereas transient oligomers may dissociate during the patching or immunoprecipitation steps (34). The mode of interaction between the BMP receptors in the homomeric and heteromeric complexes (stable versus transient interactions on the time scale of the FRAP experiments) can be assessed by the combination of IgG-mediated patching with FRAP (patch/FRAP) (34,41,42). In this approach, one receptor is patched and laterally immobilized by cross-linking with a double layer of IgGs; the effect on the lateral diffusion of a differently tagged co-expressed receptor (labeled by monovalent FabЈ fragments) is measured by FRAP (see "Experimental Procedures").…”