The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.     

Microdomains of GPI-anchored proteins in living cells revealed by crosslinking

There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane. These putative microdomains have been implicated in processes such as sorting in polarized cells and signal transduction. Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents: these complexes were thought to represent a clustered arrangement of GPI-anchored proteins. However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state. Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPI-anchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.     

Apical polarity of N-CAM and EMMPRIN in retinal pigment epithelium resulting from suppression of basolateral signal recognition

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM's basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.     

Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.     

Throat disorders

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.     

In vitro incorporation of GPI-anchored proteins into human erythrocytes and their fate in the membrane

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4 degrees C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4 degrees C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.     

Branch migration through DNA sequence heterology

We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.     

Functional competence of T cells in the absence of glycosylphosphatidylinositol-anchored proteins caused by T cell-specific disruption of the Pig-a gene

T lymphocytes express various glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Thy-1 and Ly-6A. However, functional contribution of GPI-anchored proteins in T cell activation is as yet poorly understood. Here we report the generation of mutant mice deficient in the expression of GPI-anchored molecules exclusively in their T cells. We established mice carrying three identically oriented lox-P sites within the Pig-a gene, which encodes a component essential for the initial step of GPI anchor biosynthesis. These mice were crossed with mice carrying the Cre recombinase gene driven by the T cell-specific p56lck proximal promoter. Offspring carrying both the lox-P-containing Pig-a gene and the Cre transgene exhibited almost complete loss of the surface expression of GPI-anchored molecules on peripheral T cells. Interestingly, those T cells deficient in GPI-anchored molecules were capable of responding to T cell receptor stimulation in vitro and in vivo. These results indicate that T cells lacking the expression of GPI-anchored molecules are functionally competent in exerting TCR-mediated immune responses.     

The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST-Rho39Tr) to the apical membrane. The targeting of GST-Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST-Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST-Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75(NTR)) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75(NTR) causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75(NTR) does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75(NTR), providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75(NTR) is not required for apical sorting of either transmembrane or secreted forms.     

VIP17/MAL, a proteolipid in apical transport vesicles

VIP17 is a proteolipid enriched in the CHAPS-insoluble complexes from MDCK cells, and a candidate component of the molecular machinery responsible for the sorting and targeting of proteins to the apical surface. Cloning and sequencing of the cDNA encoding the protein revealed that it is the canine homolog of the human and rat MAL proteins. Analysis by immunofluorescence microscopy of epitope-tagged VIP17/MAL expressed transiently in BHK cells and stably in MDCK cells revealed a perinuclear, vesicular, and plasmalemmal staining. In MDCK cells the distribution was mainly in vesicular structures in the apical cytoplasm. These and other results suggest that VIP17/MAL is an important component in vesicular trafficking cycling between the Golgi complex and the apical plasma membrane.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl-) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl- secretion by stimulating CFTR Cl- channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl- secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl- secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.     

Polarized secretion of apoA-I and apoA-II by transfected MDCK cells

Apolipoproteins (apo) are secreted preferentially from the basolateral surface of hepatocytes and enterocytes. The polarized secretion of proteins is either mediated by a protein-dependent sorting signal or by a cell-dependent default pathway. In order to determine the mechanism for the polarized secretion of apolipoproteins, we examined the secretion of apoA-I and apoA-II in transfected Madin-Darby canine kidney (MDCK) cells. Transfected MDCK cells and Caco-2 cells were grown as a polarized monolayer on tissue culture inserts, which separate an upper apical compartment from the lower basolateral compartment, and the secretion of apoA-I and apoA-II into the apical and basolateral compartments was quantitated by immunoprecipitation. Caco-2 cells almost exclusively secreted apoA-I and apoA-II basolaterally, with an apical to basolateral ratio of 18:82 for apoA-I, and 11:89 for apoA-II. In contrast, transfected MDCK cells secreted significant amounts of apoA-I and apoA-II into both compartments, but with a bias toward apical secretion and an apical to basolateral ratio of 66:34 and 68:32, respectively. The polarized secretion of MDCK cells was not due to transcytosis, diffusion, or differential recovery. As assessed by density gradient ultracentrifugation, apoA-I and apoA-II secreted from either the apical or basolateral surface were relatively lipid-poor. Overall, these results suggest that the polarized secretion of apoA-I and apoA-II does not occur by a protein-dependent sorting signal, but by a cell-dependent default pathway that leads to preferential basolateral secretion by Caco-2 cells and both apical and basolateral secretion in MDCK cells, but with a bias toward apical secretion.     

Disruption of microtubules reveals two independent apical targeting mechanisms for G-protein-coupled receptors in polarized renal epithelial cells

G-protein-coupled receptors demonstrate differing trafficking itineraries in polarized Madin-Darby canine kidney (MDCK II) cells. The alpha2A adrenergic receptor (alpha2AAR) is directly delivered to the basolateral subdomain; the A1 adenosine receptor (A1AdoR) is apically enriched in its targeting; and the alpha2BAR subtype is randomly delivered to both domains but selectively retained basolaterally (Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340-11347; Saunders, C., Keefer, J. R., Kennedy, A. P., Wells, J. N., and Limbird, L. E. (1996) J. Biol. Chem. 271, 995-1002; Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017-5024). The present studies explore the role of the polarized cytoskeleton in localization of G-protein-coupled receptors in MDCK II cells. Nocodazole or colchicine, which disrupt microtubules, did not perturb lateral localization of alpha2AR subtypes but led to a relocalization the A1AdoR to the basolateral surface, revealed by immunocytochemical and metabolic labeling strategies. Conversely, the apical component of the random delivery of alpha2BAR was not affected by these agents, suggesting microtubule-dependent and -independent apical targeting mechanisms for G-protein-coupled receptors in polarized cells. Apparent rerouting of the apically targeted A1AdoR was selective for microtubule-disrupting agents, since cytochalasin D, which disrupts actin polymerization, did not alter A1AdoR or alpha2BAR localization or targeting. These data suggest that multiple apical targeting mechanisms exist for G-protein-coupled receptors and that microtubule-disrupting agents serve as tools to probe their different trafficking mechanisms.     

Effects of NH4Cl and nocodazole on polarized fibronectin secretion vary amongst different epithelial cell types

The extracellular matrix protein fibronectin was found to be secreted by three polarized epithelial cell lines Madin-Darby canine kidney (MDCK), Caco-2 and LLC-PK1. About 54 and 46% of fibronectin was secreted from the apical and basolateral cell surfaces, respectively, in MDCK cells. In Caco-2 and LLC-PK1 cells, the majority (about 92-93%) of fibronectin secretion occurs from the basolateral cell surface, with the remaining 7-8% from the apical surface. In all three cell types, NH4Cl was found to inhibit basolateral secretion (resulting in enhanced apical secretion), while total fibronectin secretion was not significantly affected (although a delay in secretion was observed). Nocodazole reduced total fibronectin secretion to about 70% of control levels in MDCK and Caco-2 cells, with significant inhibition on secretion from both surfaces. In contrast, total fibronectin secretion was enhanced by nocodazole in LLC-PK1 cells. Furthermore, the majority of fibronectin secretion was redirected to the apical cell surface in LLC-PK1 cells. These observations demonstrate that the nature as well as the extent of the effects of NH4-Cl and nocodazole on polarized fibronectin secretion varies amongst different epithelial cell types.     

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Cell-surface engineering with GPI-anchored proteins

Protein engineering of cell surfaces is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer. This technology exploits the fact that proteins that are anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, incorporate into their surface membranes and are fully functional. By substituting 3'-mRNA end sequence of naturally GPI-anchored proteins (i.e., a sequence that contains the signals that direct GPI anchoring) for endogenous 3'-mRNA end sequence, virtually any protein of interest can be expressed as a GPI-anchored derivative. The GPI-anchored product then can be purified from transfectants and the purified protein used to "paint" any target cell. Such protein engineering or "painting" of the cell surface offers several advantages over conventional gene transfer. Among these advantages are that 1) GPI-anchored proteins can be painted onto cells that are difficult to transfect, 2) cells can be altered immediately without previous culturing, 3) the amount of protein added to the surface can be precisely controlled, and 4) multiple GPI-anchored proteins can be sequentially or concurrently inserted into the same cells. Emerging applications for the technology include its use for the analysis of complex cell-surface interactions, the engineering of antigen presenting cells, the development of cancer vaccines, and possibly the protection against graft rejection.     

Cholesterol-dependent retention of GPI-anchored proteins in endosomes

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins.     

Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.