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Physiol. 131, 14 -22.) To resolve this apparent contradiction, we investigated the binding, internalization, conformational changes, and productive uncoating of HRV2 in HeLa cells subjected to hypotonic shock and potassium depletion. This treatment led to an increase in HRV2 binding, with internalization being barely affected. The generation of C-antigenic particles requiring pH <5.6 was strongly reduced due to an elevation of the pH in endosomal compartments. However, K ؉ depletion only slightly affected de novo viral protein synthesis, suggesting that productivity of viral RNA in the cytoplasm is enhanced and thus compensates for the reduction in C-antigenic particles. The distinct steps in the entry pathway of HRV2 are thus differently influenced by potassium depletion. Viral internalization under conditions of inhibited clathrin-dependent endocytosis without the need to disturb the ionic milieu was confirmed in HeLa cells overexpressing the nonfunctional dynamin-1 mutant K44A. Unexpectedly, overexpression of dynamin-1 K44A resulted in elevated endosomal pH compared with overexpression of wild-type dynamin.

Cellular cholesterol uptake is mainly mediated by the LDL receptor (LDL-R) and the scavenger receptor family. We hypothesized that expression levels of key receptors of these families were regulated differently in placentas from IUGR pregnancies with varying degrees of severity. Third-trimester placentas from IUGR pregnancies with (IUGR-S) and without (IUGR-M) fetal hemodynamic changes and from control (AGA) pregnancies were studied. LDL-R, LDL-Rrelated protein (LRP-1), and scavenger receptor class B type I (SR-BI) mRNA and protein levels were measured. Cholesterol concentration and composition of lipoproteins were analyzed enzymatically and by lipid electrophoresis, respectively, in maternal and umbilical cord blood. LDL-R mRNA levels in IUGR-M were similar to AGA but lower (P Ͻ 0.05) in IUGR-S. In contrast, LDL-R protein was twofold (IUGR-M) and 1.8-fold (IUGR-S) higher (P Ͻ 0.05) than in the AGA group. LRP-1 mRNA and protein levels were not altered in the IUGR cases. SR-BI mRNA was unchanged in IUGR, but protein levels were lower (P Ͻ 0.05) in IUGR-S than in the other groups. Maternal plasma concentrations of LDL cholesterol were higher (P Ͻ 0.05) in the AGA group (188.5 Ϯ 23.6 mg/dl) than in the IUGR-S group (154.2 Ϯ 26.1). Electrophoretic mobility of the LDL fraction in maternal plasma demonstrated significant changes in migration toward higher values (AGA 0.95 Ϯ 0.06, IUGR-M 1.12 Ϯ 0.11, P Ͻ 0.001; IUGR-S 1.28 Ϯ 0.20, P ϭ 0.002). We conclude that LDL-R and SR-BI levels are altered in IUGR pregnancies. These differences were associated with changes in LDL, but not HDL, mobility and cholesterol concentration in maternal circulation. pregnancy; placenta; lipids; fetal growth CHOLESTEROL HAS MULTIPLE BIOLOGICAL ROLES that include its functioning as a structural membrane component, precursor for steroid synthesis, and activator of various cellular processes (42). Extensive steroid hormone synthesis in the placenta and the rapid growth and development of the fetus make pregnancy a condition of high cholesterol demand in the feto-placental unit (23). In humans, both placental tissue (39) and fetal organs (5) have the capacity for de novo cholesterol synthesis. However, the high cholesterol demand in the fetal tissues may not be fully satisfied by endogenous means. A significantly higher cholesterol concentration in the umbilical vein compared with the arteries (33) suggests transfer from maternal or placental sources to the fetus. In the third trimester of gestation, maternally-derived cholesterol reportedly contributes ϳ22-40% to the fetal cholesterol pool (14, 24). However, this remains controversial (25). The molecular mechanisms accounting for the uptake of maternal cholesterol into the placenta, mainly as lipoprotein-associated cholesterol (42), and for subsequent transfer, if any, into the fetal circulation, are as yet poorly understood.Cellular uptake of maternal low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL) is mainly mediated by two receptor familie...

Receptor-associated protein (RAP) was originally described as a 39-kDa intracellular protein copurifying with mammalian low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR). RAP has a high affinity for LRP/alpha 2MR and interferes with the receptor's ability to bind a variety of ligands. The laying hen expresses, in a tissue-specific manner, at least four different proteins which belong to the same family of receptors as LRP/alpha 2MR. Here we show that the chicken also produces RAP, so far thought to be expressed only in mammals. Studies on the interaction of recombinant human RAP with the LDL receptor family in the chicken revealed that RAP binds with high affinity to the abundant oocyte receptor for yolk precursors (OVR) as well as to the somatic cell-specific LRP/alpha 2MR. Significantly, RAP interacts with a lower affinity with the LDL receptor, but does not bind to the oocyte-specific form of LRP. Binding of RAP to OVR inhibits the interaction of the receptor with all known physiological ligands, i.e. the yolk precursors very low density lipoprotein, vitellogenin, and alpha 2-macroglobulin. In COS cells transfected with OVR, RAP is internalized and degraded in a concentration-dependent and saturable manner. Lactoferrin, another protein with a high affinity for mammalian LRP/alpha 2MR, also binds to OVR and abolishes its interaction with yolk precursors. Cross-competition experiments show that RAP and lactoferrin recognize sites different from those involved in yolk precursor binding. The availability of pure OVR and LDLR enable us to determine kinetic parameters for the binding of RAP and lactoferrin to these receptors by surface plasmon resonance. Taken together, our results strongly suggest that chicken OVR, which is easily accessible and highly abundant in growing oocytes, represents a superior system for studying mechanistic and structural aspects of the interaction of ligands and modulating proteins with members of the LDL receptor gene family.

Lipoprotein(a) (Lp(a)) is a complex of low density lipoprotein (LDL) with apolipoprotein (apo) (a). To examine the size distribution of Lp(a). plasma was separated by fast flow gel filtration and Lp(a):B complexes were determmed in the eluate by enzyme immunoassays, in which detection was performed with monoclonal antibodies specific for apoB. Lp(a):B particles displayed apparent molecular masses (M,) of 2 x 10h to at least 10 x 106. Lp(a) size isoforms differed by the expression of apoB epitopes and their interaction with cultured human skin fibroblasts. LDL was more effective in inhibiting bmding, uptake, and degradation of low M, Lp(a) than of high M, Lp(a). In contrast, Glu-plasminogen. a?-macroglobulin and tissue-type plasminogen activator were more effective in competing for the cellular degradation of high M, Lp(a) than of low M, Lp(a). Ligand blotting revealed that Lp(a) bound to the low density hpoprotein receptor. the low density lipoprotem receptor-related protein/a,-macroglobulin receptor (LRP) and to two other endosomal membrane proteins. We propose that the LDL receptor preferentially internalizes low A4, Lp(a), whereas LRP may have a role in the clearance of high A4, Lp(a).

No abstract

LR7/8B is a member of the low density lipoprotein receptor gene family that is specifically synthesized in the brain. Here we have functionally expressed in 293 cells the splice variant harboring eight ligand binding repeats (LR8B). As assessed by confocal microscopy, the expressed receptor is localized to the plasma membrane. Importantly, in cell binding experiments, we demonstrate that this protein is a receptor for activated ␣ 2 -macroglobulin. Because to date low density lipoprotein receptor-related protein (LRP) has been shown to be the only ␣ 2 -macroglobulin receptor in brain, we became interested in the expression pattern of both proteins at the cellular level in the brain. LR7/8B is expressed in large neurons and Purkinje cells of the cerebellum and in cells constituting brain barrier systems such as the epithelial cells of the choroid plexus, the arachnoidea, and the endothelium of penetrating blood vessels. Anti-LR7/8B antibody stains the plasma membrane, dendrites, and vesicular structures close to the cell membrane of neurons, especially of Purkinje cells. In contrast, LRP is present in patchy regions around large neurons and most prominently in the glomeruli of the stratum granulare of the cerebellum. This suggests that, contrary to LR7/8B, LRP is expressed in synaptic regions of the neurons; furthermore, there is a striking difference in the expression patterns of LR7/8B and LRP in the choroid plexus. Whereas LRP shows baso-lateral and apical localization in the epithelial cells, LR7/8B is restricted to the apical cell aspect facing the cerebrospinal fluid. Finally, these studies were extended to cultured primary rat neurons, where double immunofluorescence labeling with anti-LR7/8B and anti-microtubuli-associated protein 2 (MAP2) confirmed the somatodendritic expression of the receptor. Based upon these data, we propose that LR7/8B is involved in the clearance of ␣ 2 -macroglobulin⅐proteinase complexes and/or of other substrates bound to ␣ 2 -macroglobulin from the cerebrospinal fluid and from the surface of neurons.The LDL 1 receptor gene family specifies a group of highly related composite membrane proteins engaged in receptor-mediated endocytosis of a variety of independent ligands. The members of the family are, listed in the order of their discovery, LDL receptor (LDLR), LDL receptor-related protein (LRP), gp330/megalin, VLDL receptor (or LR8), LR11, apolipoprotein E receptor 2 (apoER2), and LR8B (1, 2). Common features of these proteins are structurally and functionally distinct modules that are defined by distinct exons in the corresponding genes. These modules are (i) the type A binding repeats of ϳ40 residues, each harboring 6 paired cysteines; (ii) type B repeats, also containing 6 cysteines each; (iii) modules of ϳ50 residues with a consensus tetrapeptide, Tyr-Trp-Thr-Asp (YWTD) (together with the type B repeats they constitute the epidermal growth factor precursor homology domain); (iv) a short stretch containing many serines and threonines carrying O-linked sugars (in the case of LR8...

The toxic effects of beta-amyloid (A beta) (1-42), apolipoprotein E (apoE) isoforms, and apoE/A beta complexes were studied in human SH-SY5Y neuroblastoma cells and fibroblasts using MTT reduction. In SH-SY5Y cells, A beta(1-42) gave time-dependent toxicity over 2-48 h, which was reduced by co-incubation with rabbit beta-very low density lipoproteins (beta-VLDL). Human recombinant apoE3 and E4 isoforms were also toxic by themselves and also potentiated A beta effects when used alone, but not when associated with beta-VLDL. None of the treatments were toxic to human fibroblasts. These results suggest that beta-VLDL has a protective role on A beta-induced neurotoxicity and that the status of apoE or the conformation of lipoprotein containing apoE particles may be important for determining the contribution of apoE to neurodegeneration.