Yoav I. Henis

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells

Ras–membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras–lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein–tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.

Different Routes of Bone Morphogenic Protein (BMP) Receptor Endocytosis Influence BMP Signaling

ABSTRACT Endocytosis is important for a variety of functions in eukaryotic cells, including the regulation of signaling cascades via transmembrane receptors. The internalization of bone morphogenetic protein (BMP) receptor type I (BRI) and type II (BRII) and its relation to signaling were largely unexplored. Here, we demonstrate that both receptor types undergo constitutive endocytosis via clathrin-coated pits (CCPs) but that only BRII undergoes also caveola-like internalization. Using several complementary approaches, we could show that (i) BMP-2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in nonraft regions, (ii) continuation of Smad signaling resulting in a transcriptional response requires endocytosis via the clathrin-mediated route, and (iii) BMP signaling leading to alkaline phosphatase induction initiates from receptors that fractionate into cholesterol-enriched, detergent-resistant membranes. Furthermore, we show that BRII interacts with Eps15R, a constitutive component of CCPs, and with caveolin-1, the marker protein of caveolae. Taken together, the localization of BMP receptors in distinct membrane domains is prerequisite to their taking different endocytosis routes with specific impacts on Smad-dependent and Smad-independent signaling cascades.

Individual palmitoyl residues serve distinct roles in H-ras trafficking, microlocalization, and signaling.

ABSTRACT H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

ABSTRACT The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Actomyosin-generated tension controls the molecular kinetics of focal adhesions

Focal adhesions (FAs) have key roles in the interaction of cells with the extracellular matrix (ECM) and in adhesion-mediated signaling. These dynamic, multi-protein structures sense the ECM both chemically and physically, and respond to external and internal forces by changing their size and signaling activity. However, this mechanosensitivity is still poorly understood at the molecular level. Here, we present direct evidence that actomyosin contractility regulates the molecular kinetics of FAs. We show that the molecular turnover of proteins within FAs is primarily regulated by their dissociation rate constant (koff), which is sensitive to changes in forces applied to the FA. We measured the early changes in koff values for three FA proteins (vinculin, paxillin and zyxin) upon inhibition of actomyosin-generated forces using two methods – high temporal resolution FRAP and direct measurement of FA protein dissociation in permeabilized cells. When myosin II contractility was inhibited, the koff values for all three proteins changed rapidly, in a highly protein-specific manner: dissociation of vinculin from FAs was facilitated, whereas dissociation of paxillin and zyxin was attenuated. We hypothesize that these early kinetic changes initiate FA disassembly by affecting the molecular turnover of FAs and altering their composition.

Membrane Interactions of a Constitutively Active GFP-Ki-Ras 4B and Their Role in Signaling EVIDENCE FROM LATERAL MOBILITY STUDIES

Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonistS-trans,trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306–1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was followed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites.

Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts

Lipid rafts play important roles in cellular functions through concentrating or sequestering membrane proteins. This requires proteins to differ in the stability of their interactions with lipid rafts. However, knowledge of the dynamics of membrane protein–raft interactions is lacking. We employed FRAP to measure in live cells the lateral diffusion of influenza hemagglutinin (HA) proteins that differ in raft association. This approach can detect weak interactions with rafts not detectable by biochemical methods. Wild-type (wt) HA and glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI) diffused slower than a nonraft HA mutant, but became equal to the latter after cholesterol depletion. When antigenically distinct BHA-PI and wt HA were coexpressed, aggregation of BHA-PI into immobile patches reduced wt HA diffusion rate, suggesting transient interactions with BHA-PI raft patches. Conversely, patching wt HA reduced the mobile fraction of BHA-PI, indicating stable interactions with wt HA patches. Thus, the anchoring mode determines protein–raft interaction dynamics. GPI-anchored and transmembrane proteins can share the same rafts, and different proteins can interact stably or transiently with the same raft domains.

Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies.

Fluorescence imaging of two independently labelled proteins is commonly used to determine their co‐localization in cells. Antibody‐mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co‐localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co‐localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. green‐ and red‐labelled) are co‐localized. Two methods are used to determine co‐localization. The ‘overlap’ analysis defines two objects as co‐localized if the centre of mass of one falls within the area of the other. The ‘nearest‐neighbour distance’ analysis considers two objects as co‐localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co‐patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP‐2 adaptor complexes. The approach presented is potentially applicable to studies of co‐localization by other methods (e.g. electron microscopy), and the nearest‐neighbour distance method can also be adapted to study phenomena of correlated placement.

Ras acylation, compartmentalization and signaling nanoclusters (Review).

Ras proteins have become paradigms for isoform- and compartment-specific signaling. Recent work has shown that Ras isoforms are differentially distributed within cell surface signaling nanoclusters and on endomembranous compartments. The critical feature regulating Ras protein localization and isoform-specific functions is the C-terminal hypervariable region (HVR). In this review we discuss the differential post-translational modifications and reversible targeting functions of Ras isoform HVR motifs. We describe how compartmentalized Ras signaling has specific functional consequences and how cell surface signaling nanoclusters generate precise signaling outputs.