Signal transduction by CD58: The transmembrane isoform transmits signals outside lipid rafts independently of the GPI-anchored isoform

The adhesion molecule CD58 is natively expressed in both a glycosylphosphatidylinositol (GPI)-anchored form and a transmembrane form. We previously demonstrated that the two isoforms of CD58 are differentially distributed in the cell membrane. The GPI-linked form resides in lipid rafts while the transmembrane form resides outside lipid rafts. Following cross-linking a fraction of transmembrane CD58 redistributes to lipid rafts. It has also been demonstrated that ligand binding to CD58 induces biological functions such as cytokine production and immunoglobulin isotype switching, indicating that cell–cell interactions result in CD58-mediated signal transduction. However, the signaling pathways involved in these activation processes are poorly defined. Here we show for the first time that cross-linking of CD58 induces protein tyrosine phosphorylation of BLNK, Syk and PLCγ, and activation of ERK and Akt/PKB. In addition, we studied how these signaling events relate to the distinct membrane localization of the two isoforms of CD58. We demonstrate that cross-linking of CD58 triggers signaling that is predominantly associated with transmembrane CD58 in nonraft microdomains. Moreover, signaling through transmembrane CD58 does not depend on coexpression of the GPI-linked isoform. Thus, despite the residence of its GPI-anchored isoform in lipid rafts and the translocation of a fraction of its transmembrane isoform to lipid rafts, CD58 signaling is triggered by the transmembrane isoform outside lipid rafts. These findings corroborate signaling outside lipid rafts, as opposed to the established notion that rafts function as essential platforms for signaling.     

The prion protein and lipid rafts

Prions are the causative agent of the transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrP(C)) undergoes a post-translational conformational conversion to the infectious form (PrP(Sc)). PrP(C) associates with cholesterol- and glycosphingolipid-rich lipid rafts through association of its glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and through interaction of its N-terminal region with an as yet unidentified raft associated molecule. PrP(C) resides in detergent-resistant domains that have different lipid and protein compositions to the domains occupied by another GPI-anchored protein, Thy-1. In some cells PrP(C) may endocytose through caveolae, but in neuronal cells, upon copper binding to the N-terminal octapeptide repeats, the protein translocates out of rafts into detergent-soluble regions of the plasma membrane prior to endocytosis through clathrin-coated pits. The current data suggest that the polybasic region at its N-terminus is required to engage PrP(C) with a transmembrane adaptor protein which in turn links with the clathrin endocytic machinery. PrP(C) associates in rafts with a variety of signalling molecules, including caveolin-1 and Fyn and Src tyrosine kinases. The clustering of PrP(C) triggers a range of signal transduction processes, including the recruitment of the neural cell adhesion molecule to rafts which in turn promotes neurite outgrowth. Lipid rafts appear to be involved in the conformational conversion of PrP(C) to PrP(Sc), possibly by providing a favourable environment for this process to occur and enabling disease progression.     

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.     

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.     

Critical role for lipid raft-associated Src kinases in activation of PI3K-Akt signalling

Lipid rafts are membrane microdomains distinct from caveolae, whose functions in polypeptide growth factor signalling remain unclear. Here we show that in small cell lung cancer (SCLC) cells, specific growth factor receptors such as c-Kit associate with lipid rafts and that these domains play a critical role in the activation of phosphoinositide 3-kinase (PI3K) signalling. The class IA p85/p110alpha associated with Src in lipid rafts and was activated by Src in vitro. Lipid raft integrity was essential for Src activation in response to stem cell factor (SCF) and raft disruption selectively inhibited activation of protein kinase B (PKB)/Akt in response to SCF stimulation. Moreover, inhibition of Src kinases blocked PKB/Akt activation and SCLC cell growth. The use of fibroblasts with targeted deletion of the Src family kinase genes confirmed the role of Src kinases in PKB/Akt activation by growth factor receptors. Moreover a constitutively activated mutant of Src also stimulated PI3K/Akt in lipid rafts, indicating that these microdomains play a role in oncogenic signalling. Together our data demonstrate that lipid rafts play a key role in the activation of PI3K signalling by facilitating the interaction of Src with specific PI3K isoforms.     

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.     

Compartmentation of Fyn kinase with glycosylphosphatidylinositol-anchored molecules in oligodendrocytes facilitates kinase activation during myelination.

In many cell types, glycosylphosphatidylinositol (GPI)-anchored proteins are sequestered in detergent-resistant membrane rafts. These are plasma membrane microdomains enriched in glycosphingolipids and cholesterol and are suggested to be platforms for cell signaling. Concomitant with the synthesis of myelin glycosphingolipids, maturing oligodendrocytes progressively associate GPI-anchored proteins, including the adhesion molecules NCAM 120 and F3, in rafts. Here we show that these microdomains include Fyn and Lyn kinases. Both kinases are maximally active in myelin prepared from young animals, correlating with early stages of myelination. In the rafts, Fyn kinase is tightly associated with NCAM 120 and F3. In contrast, in oligodendrocyte progenitor cells lacking rafts or in raft-free membrane domains of more mature cells, F3 does not associate with Fyn. The addition of anti-F3 antibodies to oligodendrocytes results in stimulation of Fyn kinase specifically in rafts. Compartmentation of oligodendrocyte GPI-anchored proteins in rafts is thus a prerequisite for association with Fyn, permitting kinase activation. Interaction of oligodendrocyte F3 with axonal ligands such as L1 and ensuing kinase activation may play a crucial role in initiating myelination.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells.     

Lipid rafts: signaling and sorting platforms of cells and their roles in cancer

Lipid rafts are defined as microdomains within the lipid bilayer of cellular membranes that assemble subsets of transmembrane or glycosylphosphatidylinisotol-anchored proteins and lipids (cholesterol and sphingolipids) and experimentally resist extraction in cold detergent (detergent-resistant membrane). These highly dynamic raft domains are essential in signaling processes and also form sorting platforms for targeted protein traffic. Lipid rafts are involved in protein endocytosis that occurs via caveolae or flotillin-dependent pathways. Non-constitutive protein components of rafts fluctuate dramatically in cancer with impacts on cell proliferation, signaling, protein trafficking, adhesion and apoptosis. This article focuses on the identification of candidate cancer-associated biomarkers in carcinoma cells using state-of-the-art proteomics.     

Lipid rafts prepared by different methods contain different connexin channels, but gap junctions are not lipid rafts

Cell extraction with cold nonionic detergents or alkaline carbonate prepares an insoluble membrane fraction whose buoyant density permits its flotation in discontinuous sucrose gradients. These lipid "rafts" are implicated in protein sorting and are attractive candidates as platforms that coordinate signal transduction pathways with intracellular substrates. Gap junctions form a direct molecular signaling pathway by end-to-end apposition of hemichannels containing one (homomeric) or more (heteromeric) connexin isoforms. Residency of channels composed of Cx26 and/or Cx32 in lipid rafts was assessed by membrane insolubility in alkaline carbonate or different concentrations of Triton X100, Nonidet P40 and Brij-58 nonionic detergents. Using Triton X100, insoluble raft membranes contained homomeric Cx32 channels, but Cx26-containing channels only when low detergent concentrations were used. Results were similar using Nonidet P40, except that Cx26-containing channels were excluded from raft membranes at all detergent concentrations. In contrast, homomeric Cx26 channels were enriched within Brij-58-insoluble rafts, whereas Cx32-containing channels partitioned between raft and nonraft membranes. Immunofluorescence microscopy showed prominent colocalization only of nonjunctional connexin channels with raft plasma membrane; junctional plaques were not lipid rafts. Rafts prepared by different extraction methods had considerable quantitative and qualitative differences in their lipid compositions. That functionally different nonjunctional connexin channels partition among rafts with distinct lipid compositions suggests that unpaired Cx26 and/or Cx32 channels exist in membrane domains of slightly different physicochemical character. Rafts may be involved in trafficking of plasma membrane connexin channels to gap junctions.     

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.     

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.     

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.     

Interaction of epithelial cell membrane rafts with Paracoccidioides brasiliensis leads to fungal adhesion and Src-family kinase activation

Membrane rafts are cholesterol- and sphingolipid-enriched cell membrane domains, which are ubiquitous in mammals and play an essential role in different cellular functions, including host cell-pathogen interaction. In this work, by using several approaches, we demonstrated the involvement of epithelial cell membrane rafts in adhesion process of the pathogenic fungus Paracoccidioides brasiliensis. This conclusion was supported by the localization of ganglioside GM1, a membrane raft marker, at P. brasiliensis-epithelial cell contact sites, and by the inhibition of this fungus adhesion to host cells pre-treated with cholesterol-extractor (methyl-beta-cyclodextrin, MbetaCD) or -binding (nystatin) agents. In addition, at a very early stage of P. brasiliensis-A549 cell interaction, this fungus promoted activation of Src-family kinases (SFKs) and extracellular signal-regulated kinase 1/2 (ERK1/2) of these epithelial cells. Whereas SFKs were partially responsible for activation of ERK1/2, membrane raft disruption with MbetaCD in A549 cells led to total inhibition of SFK activation. Taking together, these data indicate for the first time that epithelial cell membrane rafts are essential for P. brasiliensis adhesion and activation of cell signaling molecules.     

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion.     

Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis

The latent membrane protein 1 (LMP-1) oncoprotein of Epstein-Barr virus (EBV) is a constitutively active, CD40-like cell surface signaling protein essential for EBV-mediated human B-cell immortalization. Like ligand-activated CD40, LMP-1 activates NF-kappaB and Jun kinase signaling pathways via binding, as a constitutive oligomer, to tumor necrosis factor receptor-associated factors (TRAFs). LMP-1's lipid raft association and oligomerization have been linked to its activation of cell signaling pathways. Both oligomerization and lipid raft association require the function of LMP-1's polytopic multispanning transmembrane domain, a domain that is indispensable for LMP-1's growth-regulatory signaling activities. We have begun to address the sequence requirements of the polytopic hydrophobic transmembrane domain for LMP-1's signaling and biochemical activities. Here we report that transmembrane domains 1 and 2 are sufficient for LMP-1's lipid raft association and cytostatic activity. Transmembrane domains 1 and 2 support NF-kappaB activation, albeit less potently than does the entire polytopic transmembrane domain. Interestingly, LMP-1's first two transmembrane domains are not sufficient for oligomerization or TRAF binding. These results suggest that lipid raft association and oligomerization are mediated by distinct and separable activities of LMP-1's polytopic transmembrane domain. Additionally, lipid raft association, mediated by transmembrane domains 1 and 2, plays a significant role in LMP-1 activation, and LMP-1 can activate NF-kappaB via an oligomerization/TRAF binding-independent mechanism. To our knowledge, this is the first demonstration of an activity's being linked to individual membrane-spanning domains within LMP-1's polytopic transmembrane domain.     

Evidence for segregation of heterologous GPI-anchored proteins into separate lipid rafts within the plasma membrane

Cholesterol and glycosphingolipid-rich membrane rafts, which are rich in GPI-anchored proteins and are distinct from caveolae, are believed to serve as platforms for signal transduction events and protein recycling. GPI-anchored proteins with diverse functions as well as caveolin may be recovered in a membrane fraction insoluble in cold non-ionic detergent. This study tests for possible heterogeneity in the protein composition of the lipid rafts and detergent-insoluble membrane complexes by examining the two GPI-anchored homologous human folate receptors (FR)-alpha and -beta, the GPI-anchored human placental alkaline phosphatase (PLAP), and caveolin (control) in transfected CHO cells. Both FR and PLAP showed the equal distribution of cell-surface vs. sequestered (recycling) protein typical of GPI-proteins. Quantitative affinity purification of detergent-insoluble complexes using biotinylated folate or specific antibodies demonstrated a strong association of the homologous FR-alpha and FR-beta in the same detergent-insoluble complex and separate complexes containing either PLAP or caveolin. Immunogold localization experiments using antibody crosslinking to produce larger aggregates of GPI-anchored proteins for visualization by electron microscopy also showed a clear separation between FR- and PLAP-rich membrane microdomains. Thus, even though functionally diverse and heterologous GPI-anchored proteins are known to share endocytic and recycling vesicles, they may be segregated in distinct lipid rafts on the basis of their ecto(protein) domains facilitating clustering, compartmentalization and homotypic protein interactions.     

Connexin family members target to lipid raft domains and interact with caveolin-1

Lipid rafts are cholesterol-sphingolipid-rich microdomains that function as platforms for membrane trafficking and signal transduction. Caveolae are specialized lipid raft domains that contain the structural proteins known as the caveolins. Connexins are a family of transmembrane proteins that self-associate to form cell-cell connections known as gap junctions and that are linked to cytosolic proteins, forming a protein complex or Nexus. To determine the extent to which these intracellular compartments intersect, we have systematically evaluated whether connexins are associated with lipid rafts and caveolin-1. We show that connexin 43 (Cx43) colocalizes, cofractionates, and coimmunoprecipitates with caveolin-1. A mutational analysis of Cx43 reveals that the hypothesized PDZ- and presumptive SH2/SH3-binding domains within the Cx43 carboxyl terminus are not required for this targeting event or for its stable interaction with caveolin-1. Furthermore, Cx43 appears to interact with two distinct caveolin-1 domains, i.e., the caveolin-scaffolding domain (residues 82-101) and the C-terminal domain (135-178). We also show that other connexins (Cx32, Cx36, and Cx46) are targeted to lipid rafts, while Cx26 and Cx50 are specifically excluded from these membrane microdomains. Interestingly, recombinant coexpression of Cx26 with caveolin-1 recruits Cx26 to lipid rafts, where it colocalizes with caveolin-1. This trafficking event appears to be unique to Cx26, since the other connexins investigated in this study do not require caveolin-1 for targeting to lipid rafts. Our results provide the first evidence that connexins interact with caveolins and partition into lipid raft domains and indicate that these interactions are connexin specific.