Abstract
We have identified, by a combination of ligand, 45Ca2+, and immunoblotting, two large membrane proteins akin to the mammalian so-called low density lipoprotein (LDL) receptor-related protein (LRP) in chicken tissues. LRP has thus far been demonstrated only in mammalian species where it is thought to act as a receptor for proteinase-alpha 2-macroglobulin complexes and/or chylomicron remnants, lipoproteins not produced in birds. One of the chicken LRPs was demonstrated in liver, and has the same apparent Mr and hallmark biochemical properties as rat liver LRP. The other chicken LRP is smaller (approximately 380 kDa) and is expressed in ovarian follicles, but is undetectable in liver. Immunological analysis demonstrated a lack of cross-reactivity between the two LRPs, as well as between them and the previously identified chicken oocyte-specific 95-kDa receptor for the yolk precursors, very low density lipoprotein, and vitellogenin (Stifani, S., Barber, D. L., Nimpf, J., and Schneider, W. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 87, 1955-1959). As shown by ligand blotting, both chicken LRPs have the ability to interact with vitellogenin, a property they share not only with rat LRP, but also with mammalian LDL receptors. To obtain independent confirmation of the ligand blotting results, the smaller (follicular) LRP was purified and high-affinity binding of vitellogenin to it was demonstrated by a solid-phase filtration binding assay. Amino acid sequences of tryptic fragments of the smaller LRP were obtained, and its homology with human LRP demonstrated through unambiguous alignment of three fragments. Both chicken LRPs, the chicken oocyte 95-kDa receptor, as well as rat LRP, could be shown by ligand blotting to interact specifically with chicken serum alpha 2-macroglobulin. In addition, human apolipoprotein E, a ligand implicated in receptor-mediated metabolism of chylomicron remnants, also binds to the smaller chicken LRP, further emphasizing the similarities between LDL receptors and related proteins from a variety of species. In analogy to the known dichotomy of chicken LDL receptors, which is characterized by the production of the 95-kDa oocyte-specific receptor on one hand and a 130-kDa LDL receptor that is exclusively expressed in somatic cells (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139), it appears that the smaller and larger chicken LRPs also may be restricted to the oocyte and somatic cells, respectively.
Highlights
We haveidentified,by a combination of ligand, tion, human apolipoprotein E, a ligand implicated in ““Ca”, and immunoblotting, two large membrane pror-eceptor-mediatedmetabolism of chylomicronremteins akin to the mammaliasno-called low density lip- nants, binds to the smaller chicken LDL receptor-relatedprotein (LRP), further oprotein (LDL) receptor-relatedprotein(LRPi)n emphasizing the similarities between LDL receptors chicken tissues
As stasis, is the bestknown product of a group of genes specifying shown by ligand blotting, both chickenLRPs have the LDL receptors awsell as proteins related to the LrDecLeptor
Visualization of rat, bovine, andchicken LRP by detergentextracts fromlivers of estrogen-treated rats and bovine adrenal cortex,two tissues known for significant LDL receptor activity [22,32], and on chicfkoelnlicular membrane extracts (Fig. 1).As expected, VTG bound most avidly to the previously characterized 95-kDa oocyte VTG/very low density lipoprotein (VLDL) receptor ligand blotting
Summary
20 " c , followedby dialysis against 25 mM Tris-HC1,50 mM NaC1, pH 8; activated ~ z M(designated aZM)was used in all experiments. lz5ILabeled ~ z wMas prepared by the lactoperoxidase method [26]; specific radioactivities ranged from 2,000 to 5,000 cpm/ng. Immunological studies have shown that transfer to nitrocellulose, ligand blotting with radiolabeled VTG, the oocyte receptor is more closely related to themammalian human LDL, and chicken a2M, andimmunoblotting were performed. For raising antisera against the chickenooc-yte LRP (see Fig. 6), the protein was visualized by ligand blotting with "'I-VTG on nitrocellulose replicas obtained from the same SDS-polyacrylamide gel, cut out from the gel after electrophoresis under nonreducing conditions, electroeluted from the gel as described [23], and used for immunization. C. Peptide Isolation and Sequence AnalUvsis-The oocyte LRP was purified,' subjected to electrophoresis on a 4.5-189; SDS-polyacrylamide gel, and electrophoretically transferred onto a sheet of nitrocellulose with 20% methanol, 0.192 M glycine, 25 mM Tris-HCI, pH. The protein content of samples containing detergent and of lipoproteins was determined as describedpreviously [15].Normalhumanskin fibroblasts were cultured asdescribed [16]
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