Evolution of lipoprotein receptors. The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand apolipoprotein E

The laying hen expresses two different lipoprotein transport receptors in cell-specific fashion. On the one hand, a 95-kDa oocyte membrane protein mediates the uptake of the major yolk precursors, very low density lipoprotein, and vitellogenin; on the other hand, somatic cells synthesize a 130-kDa receptor that is involved in the regulation of cellular cholesterol homeostasis (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Here we show that the oocyte-specific receptor binds, in addition to the yolk precursor proteins, an apolipoprotein of mammalian origin, apolipoprotein E. Ligand blotting, a solid-phase binding assay, and antireceptor antibodies were employed to demonstrate that binding of vitellogenin, very low density lipoprotein (via apolipoprotein B), and apolipoprotein E occurs to closely related, if not identical, sites on the 95-kDa oocyte receptor. The binding properties of lipovitellin, which harbors the receptor recognition site of vitellogenin, are analogous to those of apolipoprotein E: both require association with lipid for expression of functional receptor binding. The ligand specificity of the avian oocyte lipoprotein receptor supports the hypothesis that vitellogenin, which has evolved in oviparous species, represents a counterpart to mammalian apolipoprotein E.     

The receptor for yolk lipoprotein deposition in the chicken oocyte.

The final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically synthesized yolk precursor proteins from the plasma. The two major yolk-forming components, very low density lipoprotein (VLDL) and vitellogenin (VTG), have been shown to interact with a 95-kDa protein present in detergent extracts of ovarian membranes; this protein is absent in hens of a mutant nonlaying chicken strain (Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398). Here, we have purified the 95-kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor-mediated endocytosis by ultrastructural immunolocalization, structural, and functional studies. The receptor was visualized exclusively in the oocyte proper and was absent from somatic cells, in agreement with the previously reported expression of two different lipoprotein receptors in somatic cells and oocytes, respectively, of laying hens (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Amino acid sequences of tryptic fragments of the oocyte receptor were obtained, and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains. In particular, the chicken receptor's internalization sequence, Phe-Asp-Asn-Pro-Val-Tyr, is identical with that in low density lipoprotein receptors from mammals as well as Xenopus laevis. The ligand-binding properties, specificity, and kinetic parameters of the oocyte receptor were characterized in filtration assays employing pure ligands and receptor. In conjunction with ligand-blotting experiments following limited protease digestion of the receptor, the binding assay data suggest that VTG recognizes a substructure of the VLDL-binding site. These studies establish that a cell-specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays a key role in control of oocyte growth.     

Chicken oocytes and fibroblasts express different apolipoproteins-B-specific receptors

We have previously characterized a 95-kDa plasma membrane receptor for low and very low density lipoproteins in chicken oocytes (George, R., Barber, D. L., and Schneider, W. J. (1987) J. Biol. Chem. 262, 16838-16847). We now report that somatic cells of chickens, such as fibroblasts, express a different receptor for these lipoproteins. This receptor has a Mr of 130,000 and is part of a regulatory system for cholesterol homeostasis analogous to the low density lipoprotein receptor pathway in mammalian cells. Oocytes produce only the 95-kDa receptor, while fibroblasts synthesize exclusively the 130-kDa receptor. In addition to their different Mr values, another distinctive feature of the two proteins was revealed by ligand blotting experiments: the oocyte receptor bound rabbit beta-VLDL (a class of apolipoprotein-B and -E containing lipoprotein particles), whereas the fibroblast receptor did not. Furthermore, polyclonal rabbit antibodies that recognize the oocyte 95-kDa receptor failed to cross-react with the 130-kDa protein on fibroblasts [corrected]. We suggest that different receptors have evolved in the chicken in order to facilitate the deposition of lipids into oocytes (i.e. yolk formation) with concomitant maintenance of cholesterol homeostasis in extraoocytic tissues.     

A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) consists of two polypeptides, 515 and 85 kDa, that are noncovalently associated. A 39-kDa polypeptide, termed the receptor-associated protein (RAP), interacts with the 515-kDa subunit after biosynthesis of these molecules and remains associated on the cell surface. This molecule regulates ligand binding of alpha 2MR/LRP (Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238). Titration and binding studies indicate that RAP binds to two equivalent binding sites on alpha 2MR/LRP, with a KD of 14 nM. Heterologous ligand displacement experiments demonstrated that RAP completely inhibits the binding of 125I-activated alpha 2M to human fibroblasts and to the purified alpha 2MR/LRP, with a Ki of 23 and 26 nM, respectively. A direct correlation between the degree of binding of RAP to the receptor and the degree of ligand inhibition was observed, indicating that as the RAP binding sites are saturated, alpha 2MR/LRP loses its ability to bind ligands. Thus, the amount of RAP bound to alpha 2MR/LRP dictates the level of receptor activity. A model is proposed in which alpha 2MR/LRP contains multiple ligand binding sites, each regulated by a separate RAP site.     

A novel sterol-regulated surface protein on chicken fibroblasts

In the laying hen, two different receptors for apolipoprotein B (apoB)-containing lipoproteins are expressed on somatic cells and oocytes, respectively. The somatic protein has an apparent Mr of 130,000, while the oocyte receptor is a 95-kDa protein (1989. K. Hayashi, J. Nimpf, and W. J. Schneider, J. Biol. Chem. 264:3131-3139). In order to investigate the yet unresolved relationship between these two proteins, we applied immunoblotting with anti-receptor antibodies to extracts of oocytes and chicken embryo fibroblasts. IgG fractions that recognize the 95-kDa oocyte receptor did not cross-react with the somatic receptor; however, chicken fibroblasts as well as ovarian granulosa cells that had been exposed to sterols (cholesterol and 25-OH-cholesterol) or low density lipoprotein (LDL) were shown to express a novel immunoreactive protein with an apparent Mr of 110,000. This protein is localized on the cell surface, and is unable to bind apoB-containing lipoproteins. The formation of the 110-kDa protein in fibroblasts is induced in time- and concentration-dependent fashion by sterols, concomitant with a progressive decrease in the amount of functional 130-kDa receptor protein. Following its induction, exposure of cells to LDL, but not to high density lipoprotein, caused the disappearance of the immunoreactive protein. Furthermore, the production of the 110-kDa protein did not require protein synthesis. These data are compatible with the notion that this novel receptor-related, nonfunctional protein is a truncated intermediate in the degradation pathway for the 130-kDa apoB receptor, and that the truncation generates antigenic epitope(s) shared by the 95-kDa oocyte receptor and the 110-kDa protein, but not expressed on the somatic receptor.     

LDL and cAMP cooperate to regulate the functional expression of the LRP in rat ovarian granulosa cells

Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human hLDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, alpha2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.     

Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and gp330, two members of the low density lipoprotein receptor gene family, share a multitude of cysteine-rich repeats. LRP has been shown to act as an endocytosis-mediating receptor for several ligands, including protease-antiprotease complexes and plasma lipoproteins. The former include alpha 2-macroglobulin-protease complexes and plasminogen activator inhibitor-activator complexes. The latter include chylomicron remnant-like particles designated beta-very low density lipoproteins (beta-VLDL) complexed with apoprotein E or lipoprotein lipase. The binding specificity of gp330 is unknown. In the current studies we show that gp330 from rat kidney membranes binds several of these ligands on nitrocellulose blots. We also show that both LRP and gp330 bind an additional ligand, bovine lactoferrin, which is known to inhibit the hepatic clearance of chylomicron remnants. Lactoferrin blocked the LRP-dependent stimulation of cholesteryl ester synthesis in cultured human fibroblasts elicited by apoprotein E-beta-VLDL or lipoprotein lipase-beta-VLDL complexes. Cross-competition experiments in fibroblasts showed that the multiple ligands recognize at least three distinct, but partially overlapping sites on the LRP molecule. Binding of all ligands to LRP and gp330 was inhibited by the 39-kDa protein, which co-purifies with the two receptors, suggesting that the 39-kDa protein is a universal regulator of ligand binding to both receptors. The correlation of the inhibitory effects of lactoferrin in vivo and in vitro support the notion that LRP functions as a chylomicron remnant receptor in liver. LRP and gp330 share a multiplicity of binding sites, and both may function as endocytosis-mediating receptors for a large number of ligands in different organs.     

Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin I

Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.     

Lipoprotein receptors in extraembryonic tissues of the chicken

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo.     

Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.

Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.     

Platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation of the low density lipoprotein receptor-related protein (LRP). Evidence for integrated co-receptor function betwenn LRP and the PDGF

The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507-15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor.     

Oocytes from the mutant restricted ovulator hen lack receptor for very low density lipoprotein

Hens of the "Restricted Ovulator" (R/O) chicken strain are characterized by the absence of egg-laying and concomitant severe hyperlipidemia due to a single gene defect (Ho, K. J., Lawrence, W. D., Lewis, L. A., Liu, L. B., and Taylor, C. B. (1974) Arch. Pathol. 98, 161-172). However, the underlying biochemical defect has not been identified. Previous studies on receptor-mediated growth of chicken oocytes have led to the characterization of a 95-kDa oocyte plasma membrane receptor that binds very low density lipoproteins (VLDL) (George, R., Barber, D. L., and Schneider, W. J. (1987) J. Biol. Chem. 262, 16838-16847). The current experiments demonstrate the absence of this receptor from R/O oocytes. Ligand binding experiments showed that ovarian membranes from mutant hens failed to display high affinity, saturable, and specific binding of 125I-VLDL. Ligand blotting with 125I-VLDL and Western blotting with polyclonal anti-receptor antibodies visualized the 95-kDa receptor in normal oocytes, but R/O ovarian membranes were devoid of any cross-reactive protein. Finally, plasma clearance of intravenously injected 125I-VLDL was dramatically impaired in R/O in comparison to normal hens, with a concomitant decrease in the radioactivity accumulating in R/O oocytes. These data strongly suggest that the absence of the 95-kDa receptor for VLDL from oocytes is responsible for the R/O phenotype, and that the receptor not only binds VLDL, but also mediates its uptake. This animal model provides a powerful tool for investigations of receptor-mediated growth of chicken oocytes and for the elucidation of regulatory mechanisms in lipid and lipoprotein metabolism of laying hens.     

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.     

Low binding capacity and altered O-linked glycosylation of low density lipoprotein receptor in a monensin-resistant mutant of Chinese hamster ovary cells

We have studied function and structure of the low density lipoprotein (LDL) receptors in a monensin-resistant (Monr-31) mutant isolated from Chinese hamster ovary (CHO) cells. To assay the ability of the receptor to bind LDL, we employed three methods, 125I-LDL binding to the cells at 4 degrees C, 125I-LDL binding to the receptor-phospholipid complex (Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1980) J. Biol. Chem. 255, 11442-11447), and ligand blotting (Daniel, T.O., Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1983) J. Biol. Chem. 258, 4606-4611). The LDL receptor number was similar in both CHO and Monr-31, but the binding affinity was reduced in the mutant. The semi-quantitative immunoblotting assay with an antibody directed against the COOH-terminal 14 amino acids and the ligand-blotting assay with LDL also showed that the relative steady-state level of the receptor in Monr-31 was comparable to that in CHO, whereas the binding capacity of the receptor in Monr-31 was lower than that in CHO. The precursor and degradation forms of the LDL receptors produced in the mutant cells were similar in size to those in the parental cells, but the apparent molecular mass of the mature receptor protein in sodium dodecyl sulfate-polyacrylamide gels was reduced about 5000 daltons in the mutant. These results suggest a structural change at the NH2-terminal LDL binding domain. Tests of the effects of tunicamycin, endo-alpha-N-acetylgalactosaminidase (O-glycanase), and sialidase (neuraminidase) on the molecular size of the mature receptors indicated that the reduced size of the receptor in the mutant cells resulted from altered oligosaccharide chain(s) linked to serine/threonine residues in the binding domain. We compared the molecular sizes and binding activity of human LDL receptors in several clones derived from CHO and Monr-31 cells which were transfected with human LDL receptor cDNA. The human LDL receptors produced in the transfected clones of Monr-31 were also smaller in molecular size and lower in binding capacity than those produced in the transfected clones of CHO. These results suggest that both structural and functional alteration of the LDL receptor of Monr-31 is not caused by a mutation in the structural gene of the LDL receptor but by altered processing or maturation of the receptor. The correlation of the decrease in molecular size and reduced binding capacity of the LDL receptor is discussed.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

Structure of the low-density lipoprotein receptor-related protein (LRP) promoter

The low-density Lipoprotein receptor-related protein (LRP) is a 4544-amino-acid membrane protein which closely resembles the LDL receptor in its arrangement of cysteine-rich motifs. Binding studies have suggested that one function of the molecule is as a receptor for ligands containing apolipoprotein E. We present here the sequence and structure of the promoter region of the LRP. These data show that the LRP contains no sterol regulatory element, and is not down-regulated by sterols like the LDL receptor. This lends further support to the identity of the LRP as a chylomicron remnant receptor.     

Platelet-derived growth factor mediates tyrosine phosphorylation of the cytoplasmic domain of the low Density lipoprotein receptor-related protein in caveolae

The low density lipoprotein (LDL) receptor gene family represents a class of multifunctional, endocytic cell surface receptors. Recently, roles in cellular signaling have also emerged. For instance, the very low density lipoprotein receptor (VLDLR) and the apolipoprotein receptor-2 (apoER2) function in a developmental signaling pathway that regulates the lamination of cortical layers in the brain and involves the activation of tyrosine kinases. Furthermore, the cytoplasmic domain of the LDL receptor-related protein (LRP) was found to be a substrate for the non-receptor tyrosine kinase Src, but the physiological significance of this phosphorylation event remained unknown. Here we show that tyrosine phosphorylation of LRP occurs in caveolae and involves the platelet-derived growth factor (PDGF) receptor beta and phosphoinositide 3-kinase. Receptor-associated protein, an antagonist of ligand binding to LRP, and apoE-enriched beta-VLDL, a ligand for LRP, reduce PDGF-induced tyrosine phosphorylation of the LRP cytoplasmic domain. In the accompanying paper (Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini, M., Ulery, P. G., Mikhailenko, I., Lawrence, D. L., and Strickland, D. K. (2002) J. Biol. Chem. 277, 15499-15506) Loukinova et al. further demonstrate that one form of PDGF, PDGF-BB, binds specifically to LRP and that phosphorylation of LRP requires the activation of Src family kinases. Taken together, these findings provide a biochemical basis for a cellular signaling pathway that involves apoE and LRP.     

Solubilization and characterization of the chicken oocyte vitellogenin receptor.

This paper describes the biochemical characterization of the chicken oocyte plasma-membrane receptor for one of the major lipid-carrying yolk proteins, vitellogenin (VTG). The receptor was extracted from oocyte membranes with the non-ionic detergent octyl-beta-D-glucoside and visualized by ligand blotting, with 125I-VTG as a protein with an apparent Mr of 96000, under non-reducing conditions. It exhibited high affinity for native chicken VTG (Kd 2 X 10(-7) M) but was unable to bind VTG with reductively methylated lysine residues or phosvitin (the phosphoserine-rich intracellular cleavage product of VTG). Polyclonal antibodies to the 96 kDa protein inhibited VTG binding to the receptor and were able to precipitate functional VTG-receptor activity from oocyte-membrane detergent extracts with a concomitant removal of the 96 kDa protein. Antibodies directed against the mammalian receptor for low-density lipoprotein showed cross-reactivity with the chicken oocyte VTG receptor, raising the possibility that lipoprotein receptors in birds are structurally related to those in mammalian species.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.