INTRODUCTION

Low density lipoprotein receptor (LDLR)¹ gene family members are characterized by the presence in their extracellular domains of clusters of tandemly arranged repeats, each containing six cysteines. On the cell surface, these clusters of repeats constitute the binding sites for circulating ligands; intracellularly, they are now known to be the target of a 39-kDa protein (1, 2). This protein has been identified in mammals by reproducible copurification with LDLR-related protein, a large member of the LDLR family (3-6), and hence was termed receptor-associated protein (RAP) (5). RAP is a resident protein of the endoplasmic reticulum (ER) that has been implied as chaperone of LDLR gene family proteins, which are characterized by extracellular cysteine-rich repeats (1, 7-9). Due to the high affinity and apparently specific interaction with the ligand binding domains of LDLR family members, RAP has proven useful in studies on the binding properties of receptors in vivo (9, 10), on the surface of cultured cells (11, 12), and in solid-phase assays (6, 13, 14).

Among the possible physiological ligands of mammalian LDLR family members, apolipoprotein E has particularly high affinity for certain of the receptors. It has been shown that apolipoprotein E, when overexpressed in heterologous cells, leads to aggregation of nascent receptors and that RAP can alleviate such aggregation by intracellular binding to the cysteine-rich domains (15). We are particularly interested in the LDLR gene family of the chicken (16-20) as well as in the molecular genetics of receptor-mediated lipoprotein metabolism of oviparous species (21-23). Chickens are not known to harbor an apolipoprotein E gene (24), yet the best studied member of the avian LDLR gene family, the oocyte-specific splice variant of the receptor for very low density lipoprotein and vitellogenin, termed LR8 (17, 18), has the ability to bind the mammalian apolipoprotein (25). LR8 exists in two splice variants, one containing (LR8+) and one lacking (LR8) a domain with potential for multiple O-glycosylation (the so-called "O-linked sugar domain" of the LDLR gene family) (18). These different receptor forms are expressed in cell type-specific fashion; LR8 predominates in developing germ cells and LR8+ in the accompanying somatic cells, i.e. granulosa and Sertoli cells, respectively (17, 18, 26). In ligand blots, both forms were shown to react with mammalian RAP (18). Recently, we have obtained preliminary evidence for expression of RAP or a RAP-like protein in the chicken (27). To investigate the relevance of RAP expression in correlation to that of the variant receptor forms and in regard to the information on RAP in nonmammalian species, we have initiated studies to identify and molecularly and functionally characterize the first non-amniotic RAP.

EXPERIMENTAL PROCEDURES

A 290-bp fragment of human RAP cDNA (pSA39) was produced by PCR using two synthetic oligonucleotides: 5-GACGAACTCGCCTGGAACAA (A) and 5-TGAACTTTCTCTTTGTGATG (B). The obtained fragment was ³²P-labeled using the Megaprime DNA labeling kit (Amersham) and used as probe to screen a chicken follicle gt11 cDNA library (CLONTECH). Hybridization conditions were as follows: 25% formamide, 5 × SSC, 5 × Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA), 1% SDS, and 100 µg/ml salmon sperm DNA for 20 h at 42 °C. The membranes were washed 2 × 10 min in 2 × SSC, 0.1% SDS at room temperature and 2 × 30 min in 0.1 × SSC, 0.1% SDS at 42 °C. Positive clones were amplified by PCR using gt11-specific primers, subcloned into the pGEM-T vector, and sequenced on both strands. To obtain the full-length clone, a random hexamer-primed chicken brain gt11 cDNA library (CLONTECH) was screened with a homologous 269-bp chicken RAP PCR probe (nucleotides 27-295), obtained with the primers 5-GGGGCTGGCTGACGCCAAGCGGC (C) and 5-GTGAGAGTCCTTCTTTCCGT (D) as follows: 5 × NET (0.5 M NaCl, 75 mM Tris, 20 mM EDTA), 5 × Denhardt's solution, 0.2% SDS, and 100 µg/ml salmon sperm DNA for 20 h at 65 °C. The membranes were washed 2 × 30 min in 2 × NET, 0.2% SDS at 65 °C.

Antiserum against chicken RAP was prepared against a synthetic peptide corresponding to 16 residues (101-116) of the deduced amino acid sequence of the cloned cDNA for chicken RAP. The peptide was coupled to keyhole limpet hemocyanin (28) and used for immunization of female New Zealand White rabbits as described (29). Antiserum against LR8 was prepared against the purified receptor as described (30). Isolated granulosa cell sheets (31) were attached to adhesion slides (Bio-Rad) and fixed in methanol:acetone (1:1) for 5 min at 20 °C. Alternatively, fixation was performed in 4% formaldehyde in PBS for 20 min at 23 °C, followed by incubation in 50 mM NH₄Cl for 15 min and 0.1% Triton X-100 for 5 min. Samples were then blocked in PBS containing 0.2% gelatine for 30 min at room temperature and incubated with a 1:100 dilution of antisera in PBS/gelatine for 60-120 min at room temperature. After several washes in PBS, samples were incubated with goat anti-rabbit IgG conjugated to BODIPY FL (Molecular Probes, Leiden, Netherlands) for 60 min at room temperature. Following several washes, slides were incubated in PBS containing 0.1 µg/ml propidium iodide for 10 min, washed in distilled water, and embedded in Moviol (Hoechst, Frankfurt, Germany). Samples were viewed in a Zeiss Axiophot microscope and an MRC 600 laser-scanning microscope (Bio-Rad) equipped with a Krypton laser and a double channel filter set.

Triton X-100 extracts from membrane fractions of chicken tissues were prepared as described previously (32). Crude Triton X-100 extract from chicken testes, follicles, and brain were prepared by homogenizing the tissue with an Ultra Turrax T25 homogenizer for 1 min in ice-cold solubilization buffer (4 ml/g, wet weight) containing 200 mM Tris-maleate, pH 6.5, 2 mM CaCl₂, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µM leupeptin, and 1% Triton X-100. The extraction mixture was kept on ice for 10 min and then centrifuged at 300,000 × g for 40 min at 4 °C. The resulting supernatant was stored in aliquots at 20 °C until used for immunoblotting and ligand blotting. Protein concentrations were determined by the method of Lowry et al. (33).

One-dimensional 4-20% gradient SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (34) using a minigel system (Mini-Protean^TM II Slab Cell, Bio-Rad). Samples were prepared either in the absence (nonreducing conditions) or in the presence of 5 mM dithiothreitol with heating (reducing conditions). Electrophoresis was performed at 180 V for 1 h in the presence of broad range M_r standards (Bio-Rad). Electrophoretic transfer of the proteins to nitrocellulose membrane (Hybond-C, Amersham) was performed in transfer buffer (26 mM Tris, 192 mM glycine, 20% methanol) for 1 h at 200 mA, on ice, using the Bio-Rad Mini Transblot system. The transfer was checked by staining with 0.2% Ponceau S in 3% (w/v) trichloroacetic acid and destaining with water. Western blotting was performed using specific rabbit antibodies at the concentrations indicated in the figure legends, followed by protein A-horseradish peroxidase (1:5000, Sigma) and the chemiluminescence detection method (ECL system, DuPont NEN).

COS-7 cells were transfected with chRAP ligated into a pBKCMV (Stratagene) expression plasmid using Lipofectin^TM Reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. Positive transfectants were selected by incubating the COS-7 cells in G418 (1.2 mg/ml).

Combined Ligand and Western Blotting of Chicken RAP Bound to LR8

Triton X-100 extract (200 µg of protein) obtained from COS-7 cells expressing chicken RAP or from control cells were incubated for 1 h on nitrocellulose strips containing SDS-PAGE-separated oocyte membrane proteins in TBS (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, and 2 mM CaCl₂) containing 5% skim milk and 0.1% Tween (blocking buffer). The strips were washed for 3 × 20 min with TBS containing 0.1% Tween (washing buffer) and then incubated in blocking buffer with our anti-chicken RAP antiserum (1:500). After washing for 3 × 20 min, the primary antibody was detected with protein A-horseradish peroxidase (1:5000) and the chemiluminescence detection method as described above.

For Northern blotting, total RNA (20 µg) prepared from various tissues of female and male chickens was denatured using glyoxal and dimethyl sulfoxide, separated by electrophoresis on a 1.25% agarose gel, and blotted onto Hybond N⁺ nylon membrane (Amersham) using standard methods (35). The above described 269-bp chicken RAP cDNA fragment was labeled with ³²P using the Megaprime DNA labeling kit and used as probe. The membrane was hybridized at 65 °C in 10 mg/ml BSA, 70 mg/ml SDS, 0.5 M sodium phosphate buffer (pH 6.8), 1 mM EDTA (pH 8), and the ³²P-labeled DNA probe. Washing was performed at 65 °C in 5 mg/ml BSA, 50 mg/ml SDS, 40 mM sodium phosphate buffer (pH 6.8), and 1 mM EDTA and then in 10 mg/ml SDS, 40 mM sodium phosphate buffer (pH 6.8), and 1 mM EDTA. The Hybond filter was exposed to Reflection^TM film (DuPont NEN) with intensifying screens at 80 °C.

Tissue sections from testes (>24-week-old rooster) and follicles (adult hens) were prepared for in situ hybridization as described earlier (18, 26). The sections were hybridized overnight at 45 °C with prehybridization solution containing 10% dextran sulfate and ~300 pg/µl of the digoxigenin-labeled antisense or sense RNA probes. The RNA probes were prepared as follows: A 269-bp PCR fragment (27-295) prepared from the chicken RAP cDNA by PCR amplification using the primers C (sense), and D (antisense) was subcloned into the pGEM-T vector (Promega). The purified plasmid was linearized, and the RNA probe was prepared and labeled with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase (DIG RNA labeling kit (SP6/T7)) according to the manufacturer's recommendations (Boehringer Mannheim). The slides were then washed 3 × 10 min with 0.2 × SSC and 2 × 10 min with 0.1 × SSC at 50 °C. After washing, the slides were prepared for immunodetection by incubating them in 150 mM NaCl, 100 mM Tris, pH 7.5 (buffer A) containing 3% normal goat serum and 1% BSA for 30 min at room temperature. The slides were then exposed to anti-DIG-AP Fab fragments (Boehringer Mannheim) (1:500 dilution) in the same buffer for 2 h at room temperature and extensively washed with buffer A and 100 mM NaCl, 100 mM Tris, pH 9.5, and 50 mM MgCl₂ (buffer B). The antibody containing alkaline phosphatase was then detected by incubating the slides overnight with a precipitating BM purple AP substrate (Boehringer Mannheim). The reaction was stopped by incubating the slides in 10 mM Tris, 1 mM EDTA, pH 8.0, and mounting them in Aquamount (BDH, Poole). Photographs were taken with a Zeiss Axiovert 10 light microscope.

RESULTS

A full-length chRAP cDNA was isolated from a brain gt11 cDNA library by homology screening. The 1497-bp cDNA specifies an open reading frame of 1044 bp and a 3-untranslated region of 453 bp (Fig. 1). The 348-residue protein deduced from the cDNA contained a putative signal sequence of 21 residues spanning from the initiator methionine to the cleavage site, which was assigned according to von Heijne (36) to Ala/Ser (see Fig. 1). The mature protein consists of 327 amino acids with a calculated M_r of 38,654. Comparison of the avian protein sequence with those of RAPs from man, mouse, and rat (Fig. 3) revealed identities of 65, 63, and 58%, respectively. Analysis of the primary sequence showed that chRAP contains a single potential site for N-glycosylation, as well as the highly conserved carboxyl-terminal tetrapeptide, His-Asn-Glu-Leu (HNEL, Fig. 1), previously shown to serve as an ER retention signal for human RAP (1). Indeed, determination of the subcellular distribution of chRAP in ovarian granulosa cells by immunofluorescence (Fig. 2) revealed a typical ER staining pattern with highest levels in the perinuclear region (panels A and B). The granulosa cell "sheets" were obtained ex vivo from ovarian follicles (32) and displayed the characteristic coherent epitheloid phenotype. For comparison, we also analyzed the distribution of the cell surface receptor LR8+ (Ref. 18 and discussed below), a member of the LDL receptor gene family, in these cells (Fig. 2C); highest steady state levels of the receptor are found in the cell periphery.

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In chRAP, the region immediately preceding the retention signal tetrapeptide HNEL contains an additional residue, Gln, and is different from those in the known mammalian homologues, in all of which it is Arg-Ala-Arg. The degree of identity of the avian RAP with its mammalian homologues appears lower in the central repeat domain (Fig. 3). Upon close inspection of the chicken RAP sequence, we found nine tetrapeptides consisting of three or four basic residues and zero or one hydrophobic amino acid. Interestingly, only four of the nine tri- or tetrabasic peptides in chicken RAP are conserved in the mammalian homologues (Fig. 3). RAP has been suggested to contain an internal triplication of approximately 100 residues (1, 2). Such triplication is clearly identifiable in chRAP, in that each repeat not only contains three of the nine basic tetrapeptides but also the sequences Lys-Asp-Glu-Leu (KDEL, residues 56-59; in repeat 1), His-Arg-Glu-Leu (HREL, residues 190-193; repeat 2), and the above-mentioned HNEL at the carboxyl terminus (i.e. the end of repeat 3). Finally, Pietromonaco et al. (37) noticed that the primary sequences surrounding each of the five tryptophanes in mammalian RAPs show conservation; this is true also for the five Trp residues (residues 31, 61, 124, 142, and 230, Fig. 1) in chicken RAP.

In light of RAP's proposed function as chaperone for members of the LDLR family (1, 8, 15), it was of interest to determine the sites of RAP expression in the chicken. The laying hen expresses several members of the LDLR gene family, as well as splice variants thereof (17, 18), in cell-specific fashion (17, 18, 26). As revealed by Northern blot analysis (Fig. 4), RAP expression was ubiquitous, although the levels varied widely between tissues (equal mRNA loading has previously been shown by hybridization for glyceraldehyde-3-phosphate dehydrogenase, see Fig. 2 in Novak et al. (19)). Of the tissues investigated, lung and kidney contained the highest levels, followed by testes, brain, uropygial gland, adrenals, granulosa cells (obtained from ovarian follicles as "granulosa sheet," "Gs" in Fig. 4; see also Fig. 2), ovary (different stages of follicles), and liver. The expression of RAP in cultured chicken embryo fibroblasts was higher when they were maintained in complete serum supplemented with 4 µg/ml OH-cholesterol (fibroblast/fetal calf serum) than when they had been exposed to lipoprotein-depleted serum in the presence of 2 µg/ml mevinolin (fibroblast/lipoprotein-deficient serum), a condition that leads to induction of LDLR, to which RAP binds with low affinity (38). In any case, RAP levels in cultured fibroblasts were low compared with levels found in tissues, with the possible exception of the liver.

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The levels of RAP protein, determined by Western blotting with a polyclonal rabbit anti-chicken RAP peptide antibody (see "Experimental Procedures") in several tissues confirmed its ubiquitous expression (Fig. 5A). Agreement between the transcript and protein data was generally good, with protein levels possibly lower and higher than expected in lung and liver, respectively. An interesting finding relates to the expression of RAP in the gonads of the chicken (Fig. 5, B and C). We have previously shown that the expression of a splice variant of the chicken LDLR homologue termed LR8+ (18) decreases during testicular maturation (26) and increases in granulosa cells during oocyte growth (cf. Fig. 2). LR8+ is undetectable in ejaculated sperm (26), in oocytes following ovulation, and in eggs.² Here, we found that the levels of RAP parallel that of LR8+, in that RAP levels were much higher in immature testes than in mature animals (Fig. 5). Also, levels of RAP in ovarian follicles that had not entered the rapid growth phase yet ("large white," diameter 4-5 mm, in Fig. 5C) were much lower than those in the second largest follicle ("follicle2"; diameter, 2 cm). Furthermore, RAP was undetectable in sperm and egg.

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The expression of the smaller receptor splice variant, LR8, which lacks a serine/threonine-rich domain, the so-called "O-linked sugar domain," is specific for the germ cells of the chicken (17, 26) and increases during sperm maturation (26) and oocyte growth, respectively.² Thus, in testes, changes in RAP expression levels are inverse to those of LR8, but at first sight did not appear to do so in the ovary (i.e. higher levels of RAP in larger follicles, Fig. 5C). To address this point in more detail, we determined the cellular sites of RAP expression in ovarian follicles and testis by in situ hybridization analysis on ovarian (Fig. 6, A and B) and testicular (Fig. 6, C and D) sections. In follicles the granulosa cells, and in testes the Sertoli cells contained by far the highest levels of RAP. While in the female gonads, the clear-cut cellular architecture allows unambiguous identification of the granulosa cells, i.e. the cell layer juxtaposed to the oocyte, within the seminiferous tubules of the testis, it is more difficult to distinguish somatic cells and maturing spermatoids. Nevertheless, close inspection reveals that in both the male and female gonads, expression of RAP, which binds to both LR8+ and LR8 (18, 26), in the germ cell-supporting somatic cells (which express LR8+) prevails over that in the germ cells (which express LR8).

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To obtain functional chicken RAP and to initiate studies on the possible role of RAP in the biosynthesis of the two chicken LR8 splice variant forms (17, 18), we have generated a mammalian cell line that stably overexpresses chicken RAP. From the results in Fig. 7, it is obvious that these cells express high levels of chicken RAP, identified as a 39-kDa protein by Western blotting in the transformed cells, but not in control cells, with antibodies raised against a synthetic peptide derived from the sequence of the cloned cDNA. Under these conditions, endogenous simian RAP did not cross-react with our anti-peptide antibody. However, the antibody recognized a COS-7 cell membrane protein doublet of about 100-105 kDa, which may represent endogeneous simian LDLR homologues (27).

DTT

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The high level of chicken RAP expression in these cells allowed us to directly, i.e. without prior purification, test for biological activity of the recombinant protein (Fig. 8). SDS-PAGE-separated membrane proteins of chicken ovarian extracts were transferred to nitrocellulose membranes, which were subsequently incubated with extracts prepared from control or chicken RAP-expressing COS-7 cells. Chicken RAP bound to proteins in the ovarian extract was then detected by Western blotting with our rabbit anti-chicken RAP antibody. As Fig. 8 demonstrates, chicken RAP in extracts of expressor COS-7 cells is able to bind to the 95-kDa LR8 (lane 1). Incubation with extracts of control cells (lane 2) reveal a much weaker 95-kDa signal, which is possibly due to the cross-reactivity of anti-RAP with endogeneous receptor(s) (Ref. 27; cf. Fig. 8, lane 3, and Fig. 7). Lane 4 of Fig. 8 shows an immunoblot with an antibody directed against the carboxyl-terminal 14 residues of LR8 (17) for comparison. Taken together, the results from this cell extract indirect ligand blotting procedure (EXLBlot) demonstrate that chicken RAP expressed in simian cells is an active in vitro ligand and that the chicken RAP/COS cell system will be useful to delineate the possible role of RAP in differential intracellular receptor splice variant targeting (17, 18).

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DISCUSSION

The structural conservation of RAP in a nonamniote is compatible with important functions of the 39-kDa protein in eukaryotic cells. While a role of RAP as chaperone for members of the LDLR family seems established, other functions seem plausible, particularly in the light of our current and recent findings (20) as well as those of others (14, 39), as discussed below. Collectively, the data suggest that RAP interacts with nascent cysteine-rich polypeptide chains at a point in the biosynthetic pathway of receptors where interaction with endogeneous ligands may otherwise block the correct onward processing (2, 9, 15). This notion is based on the known competitive displacement of ligands of the LDLR family by intracellular RAP, believed to bind to the so-called LDLR binding repeats, which are six cysteine-containing subdomains of ~40 amino acids each, clusters of which constitute the ligand binding domains of LDLR family members (40). In addition or alternatively, RAP plays an important role in receptor folding itself, as shown, e.g. by the reduction of intracellular aggregation of soluble minireceptor forms of LDLR-related protein (8). In fact, recently, Obermoeller et al. (2) reported that the triplicate repeats in RAP can perform differential functions, with only the carboxyl-terminal (third) repeat able to promote folding and secretion of soluble LDLR-related protein minireceptors (2).

Our present data and the finding that a non-LDLR family member, gp95/sortilin (39), and a receptor containing cysteine-rich repeats other than LDLR ligand binding repeats (20, 41, 42) can bind RAP are compatible with a role beyond its proposed functions as chaperone in the processing of LDLR family members and as inhibitor of premature ligand interaction with the receptors. Notably, the receptor termed LR11 by us (20, 42) and gp95/sortilin (39) contain domains that show sequence homology to segments of yeast Vps10p, the sorting receptor for soluble vacuolar carboxypeptidase Y (43). Both LR11 and gp95/sortilin have been purified from brain extracts by affinity chromatography on immobilized RAP (39, 42). Inasmuch as gp95/sortilin does not contain LDLR ligand binding repeats (six cysteines each) but Vps10p domains, which are characterized by 10 totally conserved cysteines (20, 39, 41, 42), it is reasonable to assume that the interaction between RAP and these proteins occurs via the cysteine-rich Vps10p domain(s). Thus, at least in vitro, interaction of receptors with RAP does not depend solely on the presence of six-cysteine repeats. In our chRAP-expressing COS cells, we have obtained preliminary evidence in [³⁵S]cysteine labeling experiments for increased secretion of cysteine-containing proteins when compared with control cells (data not shown). Thus, RAP may have a more general role in escorting proteins with domains containing (even numbered) cysteines requiring correct pairing, in particular when the cells co-synthesize ligand(s).

Another point to consider is the expression of splice variant forms of the avian very low density lipoprotein receptor homologue, LR8 (17, 18, 26). As shown here, levels of RAP correlate positively with those of the variant expressed in somatic cells, LR8+, which contains the so-called O-linked sugar domain (18). Somatic cells, such as the hepatoma cell line LMH-2A and likely the granulosa cells, where high levels of RAP are found (Figs. 2, 4, and 6), produce apolipoproteins and other potential ligands of the LDL receptor family (44). Thus, RAP may well be required for efficient expression of LR8+ on the surface of these cells. On the other hand, the plasma membrane expression of LR8, the germ cell-specific form lacking the O-linked sugar domain, does not seem to depend on RAP, possibly related to the fact that oocytes are not known to synthesize apolipoproteins. We have previously proposed that translocation of intracellular LR8 to the plasma membrane at onset of oocyte growth may be triggered by a specific signaling protein that interacts specifically with LR8 (17); the current data suggest that such signal is unlikely to involve RAP.

The molecular characterization of the first nonmammalian RAP revealed extensive similarity to amniotic RAPs. However, in reviewing the features of chicken RAP and published reports, we noted a discrepancy between the putative assignment of amino termini of different RAPs and the criteria of von Heijne (36). In the case of human RAP (5), amino-terminal sequencing of tryptic peptides, but not of the intact mature protein, resulted in the identification of Tyr as the amino terminus. However, this Tyr is preceded by Gly-Lys in the cDNA-derived protein sequence (Fig. 3); since trypsin might have cleaved between Lys and Tyr, and the Gly conforms much better to the von Heijne criteria, we suggest that the amino terminus of mature human RAP is Gly rather than Tyr and that of chicken RAP is Ser (Fig. 1). Another significant finding may be the fact that the three internal repeats in RAP are particularly distinct in the avian homologue, where they are characterized by triplication of potential ER retention signals and three times three tri-or tetrabasic sequences. This indicates that mammalian RAPs may have lost some of the ancestral structural features still discernible in the avian gene. We have obtained a COS-7 cell line expressing high levels of chicken RAP. Inasmuch as chicken RAP has all the structural hallmarks of hitherto known homologues, these cells should prove useful for receptor expression and processing studies. The high level of RAP in these cells has already allowed us to use crude cell extracts in a novel reverse ligand blotting procedure termed EXLBlot (Fig. 8), in which we have also obtained supporting evidence for our previous observation that RAP and certain members of the LDLR gene family share common epitope(s) (27). Here, the antibody was prepared against a synthetic peptide corresponding to residues 101-116 of chicken RAP, suggesting that receptor cross-reactive epitopes may not be limited to the region(s) of RAP previously identified in the mammalian system (27). Shared epitope recognition is, at least in part, responsible for the development in rats of the autoimmune disease, passive Heymann nephritis (27). While this immunopathological effect seems to be confined to the rat, clinical consequences of extracellular appearance of RAP in other animals cannot be excluded. At the very least, RAP bound to surface receptors would inhibit the binding and uptake of physiological ligands. This has been demonstrated in mice, which show delayed clearance of certain lipoprotein ligands as a consequence of overexpression of RAP (9, 15). We are now in a position to investigate the effects of intravenously administered homologous RAP on yolk precursor uptake into chicken oocytes, which is possibly the most dramatic LDLR family-mediated transport process.