High density lipoprotein uptake by scavenger receptor SR-BII

Scavenger receptor class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) lipids. It is unclear whether this process occurs at the cell membrane or via endocytosis. Our group previously identified an alternative mRNA splicing variant of SR-BI, named SR-BII, with an entirely different, yet highly conserved cytoplasmic C terminus. In this study we aimed to compare HDL uptake by both isoforms. Whereas SR-BI was mainly ( approximately 70%) localized on the surface of transfected Chinese hamster ovary cells, as determined by biotinylation, HDL binding at 4 degrees C, and studies of enhanced green fluorescent protein-tagged SR-BI/II fusion proteins, the majority of SR-BII ( approximately 80-90%) was expressed intracellularly. The cellular distribution of SR-BI was not affected by deletion of the C terminus, which suggests that the distinct C terminus of SR-BII is responsible for its intracellular expression. Pulse-chase experiments showed that SR-BII rapidly internalized HDL protein, whereas in the case of SR-BI most HDL protein remained surface bound. Like its ligand, SR-BII was more rapidly endocytosed compared with SR-BI. Despite more rapid HDL uptake by SR-BII than SR-BI, selective cholesteryl ether uptake was significantly lower. Relative to their levels of expression at the cell surface, however, both isoforms mediated selective uptake with similar efficiency. HDL protein that was internalized by SR-BII largely co-localized with transferrin in the endosomal recycling compartment. Within the endosomal recycling compartment of SR-BII cells, there was extensive co-localization of internalized HDL lipid and protein. These results do not support a model that selective lipid uptake by SR-BI requires receptor/ligand recycling within the cell. We conclude that SR-BII may influence cellular cholesterol trafficking and homeostasis in a manner that is distinct from SR-BI.     

Slow endocytosis of the LDL receptor-related protein 1B: implications for a novel cytoplasmic tail conformation

The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting that LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.     

17beta-Estradiol promotes the up-regulation of SR-BII in HepG2 cells and in rat livers

The scavenger receptor class B type I (SR-BI) binds to HDL and mediates the selective uptake of cholesterol esters from HDL to cells. SR-BII is an alternatively spliced product of the SR-BI gene that only differs in the C-terminal cytoplasmic domain. Previous studies with male mice demonstrated that SR-BII comprises about 12% of the total SR-BI/SR-BII present in liver. In the current studies we used a liver cell line, HepG2, and a rat estrogen replacement model to examine the effects of estrogen on the expression of SR-BII. HepG2 cells express SR-BI but not SR-BII. SR-BI/SR-BII - blocking antibodies demonstrated that HepG2 cells selectively internalize cholesterol esters in a SR-BI - dependent manner. Incubation of HepG2 cells with 10 pM of 17beta-estradiol for 12 h eliminated the expression of SR-BI and promoted the up-regulation of SR-BII. Radiolabeled HDL-binding studies demonstrated that 17beta-estradiol increased the number of HDL binding sites by 3-fold in HepG2 cells. However, 17beta-estradiol - treated cell internalized approximately 25% less cholesterol ester than vehicle-only-treated cells. The livers obtained from male rats and ovariectomized female rats contained SR-BI and a small amount of SR-BII. In contrast, the livers obtained from intact female rats and ovariectomized female rats receiving estrogen replacement contained SR-BII and a small amount of SR-BI. The amount of SR-BI and SR-BII in adrenal tissue was not affected by any of the experimental treatments.We conclude that estrogen up-regulates SR-BII in HepG2 cells and rat liver.     

The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.     

A functional YNKI motif in the short cytoplasmic tail of varicella-zoster virus glycoprotein gH mediates clathrin-dependent and antibody-independent endocytosis

The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.     

Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity

Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.     

A cytoplasmic PPPSP motif determines megalin's phosphorylation and regulates receptor's recycling and surface expression

Megalin is a large endocytic receptor expressed at the apical surface of several absorptive epithelia. It binds multiple ligands including apolipoproteins, vitamin and hormone carrier proteins and signaling molecules such as parathyroid hormone and the morphogen sonic hedgehog. An important characteristic of megalin is its high endocytic activity, which is mediated by tyrosine-based endocytic motifs within the receptor's cytoplasmic tail. This domain also harbors several putative consensus phosphorylation motifs for protein kinase (PK) C and casein kinase-II and one consensus motif for PKA and glycogen synthase kinase-3 (GSK3). Here we report that the cytoplasmic domain of megalin is constitutively phosphorylated depending on the integrity of a PPPSP motif, a putative GSK3 site, with a minor participation of the other phosphorylation motifs. Mutation of the serine residue within the PPPSP motif as well as blocking GSK3 activity, with two different inhibitors, significantly decreased the phosphorylation levels of the receptor. Both the megalin PPPAP mutant and the underphosphorylated wild-type receptor, by inhibition of GSK3 activity, were more expressed at the cell surface and more efficiently recycled, but they were not inhibited in their initial endocytosis rates. Altogether, these results show that the PPPSP motif and the GSK3 activity are critical to allow megalin phosphorylation and also negatively regulate the receptor's recycling.     

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.     

The cytoplasmic domain of the hyaluronan receptor for endocytosis (HARE) contains multiple endocytic motifs targeting coated pit-mediated internalization

The hyaluronic acid (HA) receptor for endocytosis (HARE) is the primary scavenger receptor for HA and chondroitin sulfates in mammals. The two human isoforms of HARE (full-length 315-kDa and a 190-kDa proteolytic cleavage product), which are type I single-pass membrane proteins, are highly expressed in sinusoidal endothelial cells of lymph nodes, liver, and spleen. Their identical HARE cytoplasmic domains contain four candidate AP-2/clathrin-mediated endocytic signaling motifs as follows: YSYFRI(2485), FQHF(2495), NPLY(2519), and DPF(2534) (315-HARE numbering). Stably transfected cells expressing 190-HARE(DeltaYSYFRI), 190-HARE(DeltaFQHF), or 190-HARE(DeltaNPLY) (lacking Motifs 1, 2, or 3) had decreased (125)I-HA endocytosis rates of approximately 49, approximately 39, and approximately 56%, respectively (relative to wild type). In contrast, 190-HARE(DeltaDPF) cells (lacking Motif 4) showed no change in HA endocytic rate. Deletions of motifs 1 and 2 or of 1, 2, and 4 decreased the rate of HA endocytosis by only approximately 41%. Endocytosis was approximately 95% decreased in mutants lacking all four motifs. Cells expressing a 190-HARE(Y2519A) mutant of the NPLY motif retained 85-90% of wild type endocytosis, whereas this mutation in the triple motif deletant decreased endocytosis to approximately 7% of wild type. Tyr in NPLY(2519) is thus important for endocytosis. All HARE mutants showed similar HA binding and degradation of the internalized HA, indicating that altering endocytic motifs did not affect ectodomain binding of HA or targeting of internalized HA to lysosomes. We conclude that, although NPLY may be the most important motif, it functions together with two other endocytic motifs; thus three signal sequences (YSYFRI, FQHF, and NPLY) provide redundancy to mediate coated pit targeting and endocytosis of HARE.     

Stem cell factor presentation to c-Kit. Identification of a basolateral targeting domain

Stem cell factor (also known as mast cell growth factor and kit-ligand) is a transmembrane growth factor with a highly conserved cytoplasmic domain. Basolateral membrane expression in epithelia and persistent cell surface exposure of stem cell factor are required for complete biological activity in pigmentation, fertility, learning, and hematopoiesis. Here we show by site-directed mutagenesis that the cytoplasmic domain of stem cell factor contains a monomeric leucine-dependent basolateral targeting signal. N-terminal to this motif, a cluster of acidic amino acids serves to increase the efficiency of basolateral sorting mediated by the leucine residue. Hence, basolateral targeting of stem cell factor requires a mono-leucine determinant assisted by a cluster of acidic amino acids. This mono-leucine determinant is functionally conserved in colony-stimulating factor-1, a transmembrane growth factor related to stem cell factor. Furthermore, this leucine motif is not capable of inducing endocytosis, allowing for persistent cell surface expression of stem cell factor. In contrast, the mutated cytoplasmic tail found in the stem cell factor mutant Mgf(Sl17H) induces constitutive endocytosis by a motif that is related to signals for endocytosis and lysosomal targeting. Our findings therefore present mono-leucines as a novel type of protein sorting motif for transmembrane growth factors.     

Sorting nexin 17 facilitates LRP recycling in the early endosome

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx 17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx 17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx 17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx 17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx 17 expression by short interfering RNA. We conclude that Snx 17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.     

Recognition of the tryptophan-based endocytosis signal in the neonatal Fc Receptor by the mu subunit of adaptor protein-2

Endocytosis of membrane proteins is typically mediated by signals present in their cytoplasmic domains. These signals usually contain an essential tyrosine or pair of leucine residues. Both tyrosine- and dileucine-based endocytosis signals are recognized by the adaptor complex AP-2. The best understood of these interactions occurs between the tyrosine-based motif, YXXPhi, and the mu2 subunit of AP-2. We recently reported a tryptophan-based endocytosis signal, WLSL, contained within the cytoplasmic domain of the neonatal Fc receptor. This signal resembles YXXPhi. We have investigated the mechanism by which the tryptophan-based signal is recognized. Both interaction assays in vitro and endocytosis assays in vivo show that mu2 binds the tryptophan-based signal. Furthermore, the WLSL sequence binds the same site as YXXPhi. Unlike the WXXF motif, contained in stonin 2 and other endocytic proteins, WLSL does not bind the alpha subunit of AP-2. These observations reveal a functional similarity between the tryptophan-based endocytosis signal and the YXXPhi motif, and an unexpected versatility of mu2 function.     

Identification of further important residues within the Glut4 carboxy-terminal tail which regulate subcellular trafficking

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.     

Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signaling.

We have previously shown that agonist-dependent phosphorylation of the PTH/PTHrP receptor occurs on its carboxyl-terminal tail. Using site-directed mutagenesis, phosphopeptide mapping, and direct sequencing of cyanogen bromide-cleaved fragments of phosphoreceptors, we report here that PTH-dependent phosphorylation occurs on the serine residues at positions 491, 492, 493, 495, 501, and 504, and that the serine residue at position 489 is required for phosphorylation. When these seven sites were mutated to alanine residues, the mutant receptor was no longer phosphorylated after PTH stimulation. The phosphorylation-deficient receptor, stably expressed in LLCPK-1 cells, was impaired in PTH-dependent internalization and showed an increased sensitivity to PTH stimulation; the EC(50) for PTH-stimulated cAMP accumulation was decreased by 7-fold. Furthermore, PTH stimulation of the phosphorylation-deficient PTH/PTHrP receptor caused a sustained elevation in intracellular cAMP levels. These data indicate that agonist-dependent phosphorylation of the PTH/PTHrP receptor plays an important role in receptor function.     

The inhibition of endocytosis affects HDL-lipid uptake mediated by the human scavenger receptor class B type I

The scavenger receptor SR-BI plays an important role in the hepatic clearance of HDL cholesterol and other lipids, driving reverse cholesterol transport and contributing to protection against atherosclerosis in mouse models. We characterized the role of endocytosis in lipid uptake from HDL, mediated by the human SR-BI, using a variety of approaches to inhibit endocytosis, including hypertonic shock, potassium or energy depletion and disassembly of the actin cytoskeleton. Our studies revealed that unlike mouse SR-BI, human SR-BI-mediated HDL-lipid uptake was reduced by inhibition of endocytosis. This was not dependent on the cytoplasmic C-terminus of SR-BI. Monitoring the uptake of both the protein and lipid components of HDL revealed that although overall lipid uptake was decreased, the degree of selective lipid uptake was increased. These data suggest that that endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity.     

The inhibition of endocytosis affects HDL-lipid uptake mediated by the human scavenger receptor class B type I

The scavenger receptor SR-BI plays an important role in the hepatic clearance of HDL cholesterol and other lipids, driving reverse cholesterol transport and contributing to protection against atherosclerosis in mouse models. We characterized the role of endocytosis in lipid uptake from HDL, mediated by the human SR-BI, using a variety of approaches to inhibit endocytosis, including hypertonic shock, potassium or energy depletion and disassembly of the actin cytoskeleton. Our studies revealed that unlike mouse SR-BI, human SR-BI-mediated HDL-lipid uptake was reduced by inhibition of endocytosis. This was not dependent on the cytoplasmic C-terminus of SR-BI. Monitoring the uptake of both the protein and lipid components of HDL revealed that although overall lipid uptake was decreased, the degree of selective lipid uptake was increased. These data suggest that that endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity.     

A Screen for Endocytic Motifs

Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin‐mediated endocytosis have been identified: YXXΦ, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence‐activated cell sorting (FACS)‐based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine‐like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXΦN, which is able to function in cells expressing a mutant µ2 that cannot bind YXXΦ, indicating that it is not a variation on the YXXΦ motif. Similar sequences are present in endogenous proteins, including a functional YXXXΦN (in addition to a classical YXXΦ) in cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4). Thus, the repertoire of endocytic motifs is more extensive than the three well‐characterized sorting signals.     

NPXY motifs in the beta1 integrin cytoplasmic tail are required for functional reovirus entry

Reovirus cell entry is mediated by attachment to cell surface carbohydrate and junctional adhesion molecule A (JAM-A) and internalization by beta1 integrin. The beta1 integrin cytoplasmic tail contains two NPXY motifs, which function in recruitment of adaptor proteins and clathrin for endocytosis and serve as sorting signals for internalized cargo. As reovirus infection requires disassembly in the endocytic compartment, we investigated the role of the beta1 integrin NPXY motifs in reovirus internalization. In comparison to wild-type cells (beta1+/+ cells), reovirus infectivity was significantly reduced in cells expressing mutant beta1 integrin in which the NPXY motifs were altered to NPXF (beta1+/+Y783F/Y795F cells). However, reovirus displayed equivalent binding and internalization levels following adsorption to beta1+/+ cells and beta1+/+Y783F/Y795F cells, suggesting that the NPXY motifs are essential for transport of reovirus within the endocytic pathway. Reovirus entry into beta1+/+ cells was blocked by chlorpromazine, an inhibitor of clathrin-mediated endocytosis, while entry into beta1+/+Y783F/Y795F cells was unaffected. Furthermore, virus was distributed to morphologically distinct endocytic organelles in beta1+/+ and beta1+/+Y783F/Y795F cells, providing further evidence that the beta1 integrin NPXY motifs mediate sorting of reovirus in the endocytic pathway. Thus, NPXY motifs in the beta1 integrin cytoplasmic tail are required for functional reovirus entry, which indicates a key role for these sequences in endocytosis of a pathogenic virus.     

The Rous sarcoma virus Env glycoprotein contains a highly conserved motif homologous to tyrosine-based endocytosis signals and displays an unusual internalization phenotype

The cytoplasmic domains of retroviral transmembrane (TM) glycoproteins contain conserved sequence motifs that resemble tyrosine-based (YXXO-type) endocytosis signals. We have previously described a mutant Rous sarcoma virus (RSV) Env protein, Env-mu26, with an L165R mutation in the membrane-spanning domain (MSD) of TM, that exhibited dramatically decreased steady-state surface expression (G. L. Davis and E. Hunter, J. Cell Biol. 105:1191-1203, 1987; P. B. Johnston, J. Y. Dong, and E. Hunter, Virology 206:353-361, 1995). We now demonstrate that the tyrosine of the Y(190)RKM motif in the RSV TM cytoplasmic domain is crucial for the mu26 phenotype and is part of an efficient internalization signal in the context of a mutant MSD. In contrast, despite the presence of the Y(190)RKM motif, wild-type RSV Env is constitutively internalized at a slow rate (1.1%/min) more characteristic of bulk uptake during membrane turnover than of active clustering into endocytic vesicles. The mu26 mutation and two MSD mutations that abrogate palmitoylation of TM resulted in enhanced Env endocytosis indicative of active concentration into coated pits. Surprisingly, an Env-Y190A mutant was apparently excluded from coated pits since its uptake rate of 0.3%/min was significantly below that expected for the bulk rate. We suggest that in RSV Env an inherently functional endocytosis motif is silenced by a counteracting determinant in the MSD that acts to prevent clustering of Env into endocytic vesicles. Mutations in either the cytoplasmic tail or the MSD that inactivate one of the two counteracting signals would thus render the remaining determinant dominant.     

The LOX-1 scavenger receptor cytoplasmic domain contains a transplantable endocytic motif

Oxidized low-density lipoprotein particles is a pro-atherogenic factor implicated in atherosclerotic plaque formation. The LOX-1 scavenger receptor binds OxLDL and is linked to atherosclerotic plaque initiation and progression. We tested the hypothesis that the LOX-1 cytoplasmic domain contains a transplantable signal for membrane protein endocytosis. Structural modeling of the LOX-1 cytoplasmic domain reveals that a tripeptide motif (DDL) implicated in LOX-1 endocytosis is part of a curved β-pleated sheet structure. The two aspartic acid residues within this structural model are highly solvent-accessible enabling recognition by cytosolic factor(s). A triple alanine substitution of the DDL motif within the LOX-1 scavenger receptor substantially reduced endocytosis of OxLDL. Transplantation of the LOX-1 cytoplasmic domain into a transferrin receptor reporter molecule conferred efficient endocytosis on this hybrid protein. Mutation of the DDL motif within the hybrid LOX-1-TfR protein also substantially reduced receptor-mediated endocytosis. Thus a transplantable endocytic motif within the LOX-1 cytoplasmic domain is needed to ensure efficient internalization of pro-atherogenic OxLDL particles.