(Received for publication, June 22, 1995)
From the
An abundant 95-kDa protein belonging to the low density lipoprotein receptor supergene family is essential for chicken oocyte growth by mediating the uptake of multiple plasma-borne yolk precursors. This receptor harbors at the amino terminus a cluster of eight tandemly arranged repeats typical of the ligand binding domains of members of this family and is designated low density lipoprotein receptor relative with 8 repeats (LR8). Here, we demonstrate by reverse transcriptase-polymerase chain reaction, Northern, and Western blot analyses that the chicken expresses two forms of LR8, which are generated by differential splicing of an exon encoding a serine- and threonine-rich region characteristic of LRs, termed O-linked sugar domain. The female germ cell of the chicken expresses extremely high levels of the short form of LR8 (LR8-), i.e. the 95-kDa protein; in contrast, somatic cells express lower but detectable levels of the form containing the O-linked sugar domain (LR8+). The main sites of LR8+ expression in the chicken are the heart and skeletal muscle, i.e. the same tissues where LR8 mRNAs predominate in mammals; in addition, in situ hybridization demonstrates that a significant amount of LR8+ is produced in the hen's ovarian follicular granulosa cells. We found no apparent functional difference between the two receptor forms; however, cell type-specific targeting of the multiple ligands of these receptors possibly relates to their respective expression on the cell surface.
Receptors belonging to the low density lipoprotein receptor
(LDLR) ()supergene family play key roles in both systemic
transport processes and oocyte growth in oviparous species (Schneider
and Nimpf, 1993; Schneider, 1995). One of the recently characterized
receptors in this group is a chicken protein whose established major
functions are the transport of very low density lipoprotein (VLDL),
vitellogenin,
-macroglobulin, and lactoferrin into
rapidly growing oocytes (Bujo et al., 1994, Jacobsen et
al., 1995; Hiesberger et al., 1995). This chicken
receptor, as well as its mammalian homologues (the so-called VLDL
receptors), possesses in its ligand binding domain eight tandemly
arranged complement-type repeats, each consisting of approximately 40
residues, that display a triple-disulfide bond-stabilized negatively
charged surface. Receptors with eight ligand-binding repeats have been
shown to interact with apolipoprotein (apo)B (George et al.,
1987; Nimpf et al., 1988), apoE-rich VLDL and so-called
-migrating VLDL (Hayashi et al., 1989; Takahashi et
al., 1992; Sakai et al., 1994), and the 39-kDa
receptor-associated protein (RAP) (Battey et al., 1994; Wiborg
Simonsen et al., 1994; Hiesberger et al., 1995).
Because this broad ligand specificity does not allow for unambiguous
nomenclature based on ligand definition, we here designate them LDLR
relatives with eight repeats, LR8 in short, to distinguish them from
the classical LDLRs (LR7), which harbor a cluster of seven such
repeats.
Analysis of the mutant ``restricted ovulator'' hen, characterized by lack of oocyte growth that results in severe hyperlipidemia and sterility (Ho et al., 1974; Jones et al., 1975), revealed that its oocytes neither express normal LR8 nor bind VLDL or vitellogenin (Nimpf et al., 1989; Stifani et al., 1990a). These results indicate that LR8 is essential for yolk lipoprotein deposition into oocytes, a key requirement for reproduction. In this context, the expression of LR8 in mammals is of interest. In contrast to the major site of expression in oviparous species, the ovary, mammalian LR8s are found in tissues with active metabolism of fatty acids, such as skeletal muscle, heart, adipose tissue, and brain (Takahashi et al., 1992; Gåfvels et al., 1993, 1994; Oka et al., 1994b; Webb et al., 1994; Jokinen et al., 1994). The chicken oocyte, on the other hand, avidly takes up yolk proteins including VLDL, not for immediate catabolism but rather for storage and later use as energy supply for the developing embryo.
Previously, we have identified by
Northern blotting transcripts of a size similar to that of the oocyte
LR8 in tissues of the chicken other than the ovary (Bujo et
al., 1994). In particular, low levels of cross-reactive mRNA
species were found in heart and skeletal muscle, i.e. the
major sites of expression of mammalian LR8s (Takahashi et al.,
1992; Gåfvels et al., 1993, 1994; Oka et al.,
1994b; Webb et al., 1994; Jokinen et al., 1994).
Mammalian LR8 transcripts exist in splice variant forms: the major form
is a mRNA that contains an exon coding for the so-called clustered O-linked sugar domain, but mRNAs coding for LR8 lacking that
domain have also been identified (Sakai et al., 1994; Webb et al., 1994; Jokinen et al., 1994). The sites of
expression of the different forms in mammals have not been studied in
detail. Since lack of the O-linked sugar domain is one of the
characteristics of the well characterized chicken oocyte LR8, we
considered the possibility that other somatic tissues of the chicken
may produce low levels of a differently spliced LR8 transcript.
Recently, in the course of studies on the mutation in LR8 causing the
restricted ovulator phenotype, ()we have identified such a
transcript because of its enhanced expression in mutant ovaries. Here,
we demonstrate that chickens indeed produce these two differently
spliced LR8 mRNAs, differing by 90 nucleotides, and the corresponding
proteins in cell type-specific fashion. Interestingly, we could not
detect a functional difference between the two receptor forms in
vitro. However, since only rapidly growing oocytes express on
their surface high levels of LR8 lacking the O-linked sugar
domain, cell type-specific receptor production may be important for
ligand targeting in vivo.
Figure 1: Identification of the splice variant forms of chicken LR8. A, schematic representation of chicken oocyte LR8-. The signal sequence (SS) and the following domains are shown: eight binding repeats in the ligand binding domain; the cysteine-rich repeats A, B, and C in the epidermal growth factor precursor homology domain; transmembrane domain (TM); and the cytoplasmic domain (Cyto.). Nucleotide (nt) and amino acid (a.a.) numbers (Bujo et al., 1994) are shown below and above the schematic representation, respectively. The primers P1-P4 (shortarrows) used for PCR amplification and the sizes of amplified fragments as expected from LR8- are indicated. The region of the cDNA covered by the probes used for Northern blot analysis, corresponding to the region common to LR8- and LR8+, is shown by a boldline (nucleotide number -12 to 1881). B, PCR-amplified products separated on a 1.0% agarose gel. The pair P3/P4 was used to amplify cDNA from pooled follicles (0.2-2.0 cm in diameter) as described under ``Materials and Methods.'' The main fragment of expected size (583 nucleotides, see A) and the additional longer fragment are indicated by closed and openarrowheads, respectively. The 100-Base Pair Ladder (Pharmacia) was used as size marker. C, alignment of O-linked sugar domains of LR8+ from the chicken, the rat (Jokinen et al., 1994), the mouse (Gåfvels et al., 1994), the rabbit (Takahashi et al., 1992), and man (Gåfvels et al., 1993; Sakai et al., 1994; Webb et al., 1994). Alignment was performed by the GeneWorks computer program (IntelliGenetics Inc.), and gaps were introduced to optimize alignment. The additional 30-amino acid sequence in chicken LR8+ (openarrowheads) between residues 726 and 727 of LR8- (closedarrowheads) is shown. Serine and threonine residues in the chicken protein are emphasized by asterisksabove the aligned sequences, and identical residues in all proteins are indicated by darkshading.
Thus, we performed RT-PCR and Northern blot analysis of transcripts in different chicken tissues. PCR with the primer pair P3/P2 (cf. Fig. 1A) showed that the relative levels of expression in ovary, muscle, and heart of the variants described in Fig. 1were significantly different (Fig. 2). The product corresponding to the amplified region in LR8+ (openarrow, of the expected size (375 bp)) predominates in muscle and heart (lanes2 and 3) in distinct contrast to the ovary, where LR8- (closedarrow, product of the expected size (285 bp)) is by far the major form. In fact, the ratios of intensities of the two amplified fragments (LR8-/LR8+) were similar in muscle and heart but opposite to that in ovary. These results indicate that the splice variants are expressed at vastly different levels in these major sites of LR8 expression in the chicken. Only very few, if any, RT-PCR amplifiable transcripts were found in brain, kidney, and liver (data not shown; see below).
Figure 2: Tissue-dependent expression of splice variants of LR8. mRNA from ovary (lanes1 and 4), muscle (lanes2 and 5), and heart (lanes3 and 6) were used for cDNA synthesis with (lanes1-3) or without (lanes4-6) reverse transcriptase for PCR. Amplified products using the primer pair P3/P2 (see Fig. 1A) were separated on a 1.5% agarose gel. The different splice forms, with or without O-linked sugar domain, are indicated by open and closedarrowheads, respectively. The size marker is a 100-Base Pair Ladder (Pharmacia).
To obtain further evidence for the production
of differently spliced transcripts, we next directly identified
LR8- and LR8+ mRNAs in chicken tissues by Northern blot
analysis. Previously, chicken LR8 transcripts in ovary, heart, and
muscle have been shown, with probes corresponding to regions outside
the O-linked domain, to have a size of 3.5 kb (Bujo et al., 1994). However, the current results suggest that these
3.5-kb signals might actually represent both splice variant forms
of chicken LR8. Therefore, we analyzed the original Northern blot with
the 90-bp probe specifying exactly the O-linked sugar domain
in chicken LR8+. With this probe, an expression pattern very
different from that obtained with the probe corresponding to the common
domains (Bujo et al., 1994) was obtained (Fig. 3).
Under these conditions, the
3.5-kb signal, representing
exclusively LR8+ transcripts, was detected only in muscle and
heart but not in the ovary (upon prolonged exposure, there was a faint
signal in ovary). These results reveal that there exist two forms of
LR8 mRNA in the chicken, characterized by possessing or lacking a
region coding for an O-linked sugar domain, and that their
relative levels of expression vary greatly among different tissues
and/or cells.
Figure 3: Identification of splice variants of LR8 mRNA by Northern blotting. Hybridization was performed with the 90-bp probe specific for the O-linked sugar domain of chicken LR8+. Lane1, ovary; lane2, heart; lane3, muscle; lane 4, brain; lane 5, liver; lane 6, adrenal gland; lane 7, spleen; lane 8, lung; lane 9, kidney; and lane 10, small intestine. Autoradiography was at -70 °C for 7 days with intensifying screens.
Figure 4:
Immunological identification of the splice
variant forms of LR8 protein. Membrane extracts of ovarian follicles
(0.2 µg, lane1), heart from laying hen (35
µg, lane2), and heart from rooster (35 µg, lane3) were subjected to 4.5-18%
SDS-polyacrylamide gel electrophoresis under non-reducing conditions
and processed for immunoblotting with the IgG directed against a
C-terminal peptide of chicken LR8 as described under ``Materials
and Methods.'' The signals corresponding to LR8- (the
previously described 95-kDa protein) (Bujo et al., 1994) and
to LR8+ are indicated by closed and openarrowheads, respectively. Exposure was for 10 s on
Hyperfilm-ECL.
To gain insight into possible functional differences
between the two receptor forms, we expressed them in heterologous
somatic cell systems (for the expression of LR8-, cf.
Bujo et al. (1994) and Jacobsen et al.(1995)) and
attempted to study their properties in cell surface binding studies.
However, we consistently observed vastly different degrees of surface
expression of the two forms, rendering such an approach inappropriate.
Furthermore, the very low expression of LR8+ in tissues (cf. Fig. 4) precluded analysis by binding studies with
physiological ligands. However, we already know that the best
characterized non-lipoprotein ligand of the LR family, i.e. RAP, binds to LR8- with higher affinity than lipoproteins
(Hiesberger et al., 1995). Thus, having identified the heart
as a site of co-expression of LR8- and LR8+, we used I-RAP in ligand blots (Fig. 5) on extracts from
this tissue; follicle extract served as positive control for the
binding of RAP to LR8-. Clearly, LR8+ binds RAP as well;
importantly, the ratio of intensities of the bands visualized via
binding of
I-RAP (Fig. 5) and
I-VLDL
(not shown) is equal to that detected by Western blotting (Fig. 4) or at the transcript level ( Fig. 2and Fig. 3).
Figure 5:
Binding of I-RAP to both LR8
splice variants. Chicken ovarian follicle membrane extract (lane1) and laying hen heart extract (lane2) were subjected to SDS-polyacrylamide gel
electrophoresis and ligand blot analysis using
I-labeled
RAP as described under ``Materials and Methods.'' The binding
of
I-RAP to LR8+ (openarrowhead)
and LR8- (closedarrowhead) was visualized by
autoradiography for 24 h. Numbers on the left correspond to the molecular masses (kDa) of marker
proteins.
The major site of expression of chicken LR8- is the growing oocyte (George et al., 1987; Hayashi et al., 1989; Barber et al., 1991; Shen et al., 1993; Bujo et al., 1994). Interestingly, previous in situ hybridization analysis of ovarian sections showed that receptor mRNA is concentrated in a narrow zone underlying the plasma membrane of vitellogenic oocytes (Bujo et al., 1994). The ovarian follicle preparations analyzed routinely consist of the giant oocyte as well as somatic cells, in particular granulosa cells and theca cells that surround the oocyte. To determine whether the small amount of LR8+ transcript detected in follicles ( Fig. 2and Fig. 3) was derived from any or all of the somatic cells, we performed the following experiments ( Fig. 6and Fig. 7). For analysis of cell-specific mRNAs, the follicle was mechanically dissected (Gilbert et al., 1977) to isolate the granulosa cell monolayer (lanes1) and the thecal cells (lanes2). We also analyzed mRNA isolated from undissected follicles containing oocytes at different growth stages (lanes3 and 4). The mRNAs were hybridized with the probes reacting with both LR8 transcripts or with the O-linked sugar domain-specific probe, respectively. The results of Fig. 6demonstrate that granulosa cells, as well as follicles (both 0.3 and 2 cm in diameter), contain detectable levels of LR8 transcripts; the oocyte-containing follicles clearly express high levels of LR8-, and the somatic cells express LR8+.
Figure 6:
Localization of splice variant forms of
the LR8 mRNA in chicken ovarian follicles. 2 µg of
poly(A)-RNA isolated from granulosa cell sheets (lane1), the connective tissue surrounding the
follicles (thecal layers) (lane2), and from two
undissected follicles containing oocytes of different sizes (lane3, 0.3 cm, and lane4, 2 cm in
diameter) were used for Northern blot analysis on a 1.0% agarose gel.
Hybridization was performed as described in Fig. 3B.
The left and rightpanels show the results
of hybridization with the probes corresponding to the common region
(see Fig. 1A) and the probe specific for the O-linked sugar domain, respectively.
-DNA digested by HindIII was used as size marker (in kb). Exposure time was 6 h
for the leftpanel and 5 days for the rightpanel, with intensifying
screens.
Figure 7: Localization in a chicken follicle of the LR8+ mRNA. Cryostat sections of a chicken follicle (diameter, 6 mm) were subjected to in situ hybridization with an antisense (A) and a sense (B) digoxigenin-labeled RNA probe corresponding to the O-linked sugar domain of LR8+. The hybridized probe was visualized using alkaline phosphatase-coupled goat anti-digoxigenin IgG as described under ``Materials and Methods.'' The horizontal cell layer, distinctively stained in the middle part of panelA, represents the granulosa cells (g), which separate the theca cell layer (t) from the oocyteproper (o). Bar, 10 µm.
Since granulosa cells are present in the follicle preparations (see above), these results suggested that the minute amounts of LR8+ transcript seen in lanes3 and 4, rightpanel (better visualized by prolonged exposure), are derived from these somatic cells. Direct evidence for exclusive expression of LR8+ in granulosa cells was obtained by in situ hybridization analysis of a vitellogenic follicle (Fig. 7). To this end, we used a 90-nucleotide probe corresponding exactly to the O-linked sugar domain in LR8+; the oocyte showed no reactivity with this probe. These findings are in accordance with our previous conclusion (George et al., 1987; Hayashi et al., 1989; Barber et al., 1991; Shen et al., 1993; Bujo et al., 1994) that within the follicle, it is the female germ cell that expresses LR8-.
LDL receptor family members containing a single cluster of
eight complement-type repeats co-exist with other relatives in the same
organism (Takahashi et al., 1992; Gåfvels et
al., 1993, 1994; Sakai et al., 1994; Oka et al.,
1994a, 1994b; Webb et al., 1994; Bujo et al., 1994;
Jokinen et al., 1994). Of particular interest is the emerging
notion that such co-expression cannot be required because of exclusive
ligand recognition by these receptors (Brown and Goldstein, 1986;
Strickland et al., 1990, 1991; Beisiegel et al.,
1991; Herz et al., 1991; Stifani et al., 1990b; Bu et al., 1992; Chappell et al., 1992; Huettinger et al., 1992; Orth et al., 1992; Willnow et
al., 1992; Kounnas et al., 1992, 1993; Nykjaer et
al., 1992, 1993; Battey et al., 1994; Fischer et
al., 1993; Hofer et al., 1994; Wiborg Simonsen et
al., 1994; Schneider, 1995). In fact, several ligands, such as
apoE, -migrating VLDL, and RAP are ligands for all, and others,
such as LDL and chylomicron remnants, for more than one of the known
members of the family. The family thus far includes the classical LDLR,
the so-called VLDL receptor, and the ambiguously named LDLR-related
protein/
-macroglobulin receptor(s)
(LRP/
MR) as well as glycoprotein 330/megalin (Saito et al., 1994). We feel that the extensively overlapping and
currently poorly understood ligand recognition spectra of these
receptors preclude a useful nomenclature based on ligand designation;
rather, we suggest to use the distinct structural features of these
molecules (in particular the number of complement-type repeats) for
their identification. Thus, here we designate these LDLR relatives
(LRs) as LR7 and LR8 and propose the designations LR31 to LR36 for the
large members of the family.
Mammalian LR8s are most abundantly expressed in heart, skeletal muscle, brain, and adipose tissue, but not in liver, one of the the major sites of expression of LR7 and LR31 (Takahashi et al., 1992; Gåfvels et al., 1993, 1994; Oka et al., 1994b; Webb et al., 1994; Jokinen et al., 1994). Recently, it has been reported that the human ovary also expresses LR8 (Webb et al., 1994); this is of potential significance in the light of our finding that in the chicken, LR8 expression is by far the highest in oocytes (George et al.(1987); Hayashi et al.(1989); Barber et al.(1991); Shen et al.(1993); Bujo et al.(1994) and present data).
We have now discovered that those tissues which express LR8 in mammals also express this receptor in chicken, albeit at very low levels compared to the oocytes. In the chicken, the role of LR8 as an important mediator of oocyte growth via yolk deposition has been established both by biochemical and genetic evidence (George et al., 1987; Nimpf et al., 1989; Barber et al., 1991; Stifani et al., 1990b; Shen et al., 1993). However, in mammals neither the function nor the exact site of expression of LR8 in the ovary is known.
One difference in the structures of the major LR8s in mammals and the chicken oocyte LR8, i.e. the presence and absence of the O-linked sugar domain, respectively, prompted us to investigate in detail the expression of LR8 splice variants in the egg-laying animal. The results strongly suggest that somatic cells and tissues, in particular granulosa cells, heart, and skeletal muscle express predominantly LR8+, while the oocyte is by far the major site of LR8- expression. In the context of findings in the rat (Jokinen et al., 1994), which are compatible with a role of LR8 other than in lipoprotein metabolism, we interpret the results in the laying hen as follows. Oocytic LR8- is a multifunctional receptor, which transports lipoproteins and other components (Mac Lachlan et al., 1994; Jacobsen et al., 1995) required for embryonic growth; with the exception of unique yolk precursors, this may hold true for other tissues, including the mammalian ovary. However, a definitive answer to the physiological role of LR8- in mammals cannot be provided at present. LR8+, on the other hand, is likely to perform similar functions in mammals and oviparous species, as they express this receptor in the same tissues. Again, a physiological role of LR8+, not directly related to the transport of lipoproteins, has been suggested (Jokinen et al., 1994).
It is noteworthy
that for rat and mouse LR8+, possible alternative splicing outside
the region considered here has been discovered during cloning studies
(Gåfvels et al., 1994; Jokinen et al., 1994).
Northern blot analysis in rabbit, rat, mouse, and human tissues
revealed, in addition to the transcripts corresponding to
LR8-/LR8+, transcripts of 9.5 kb in rabbit (Takahashi et
al., 1992), 9.1 kb in rat (Jokinen et al., 1994), 4.5 and
7.9 kb (Oka et al., 1994b) or 8 kb (Gåfvels et
al., 1994) in mouse, and 6.0 kb (Gåfvels et al.,
1993) or 5.2 kb (Webb et al., 1994) in human. The exact nature
of these transcripts has not been delineated; in rats, all transcripts
are coordinately regulated in response to changes in the thyroid status
(Jokinen et al., 1994). We have not detected any other but the
3.5-kb LR8 transcripts in the chicken.
In the light of our
limited knowledge of the true physiological ligand(s) of LR8s, we can
only speculate about the functional role of their O-linked
sugar domain. In this context, preliminary experiments indicate that
LR8- expressed in COS-7 or LR7-negative Chinese hamster ovary
cells leads to detectable but poor surface activity (Bujo et
al., 1994), while LR8+ is much more efficiently presented at
the cell surface (data not shown). ()Thus, due to the very
low levels of LR8+ protein in tissues, and as a result of these
vastly different levels of surface expression of LR8+ versus LR8- in transfected cells, we could only analyze binding of
the ligand with the highest affinity for the receptors (Fig. 5).
Based on current results, we consider it unlikely, but nevertheless
possible, that the ligand specificities of the two variant receptors
differ sufficiently to direct different ligands to oocytes and somatic
cells, respectively. Rather, it appears that regulation of surface
expression of the variants in different cells or tissues holds the key
to ligand targeting (Schneider, 1995). In strong support of this
notion, deletion of the O-linked sugar domain in LR7, which is
associated with very poor surface expression, brings about the clinical
symptoms of familial hypercholesterolemia (Kajinami et al.,
1988; Koivisto et al., 1992, 1993) despite unaltered ligand
recognition by the mutant protein in vitro (Davis et
al., 1986). We postulate that the chicken's female germ cell
has specific means to express high levels of LR8- on the cell
surface (George et al., 1987; Barber et al., 1991),
and that this might be a regulatory mechanism for initiating oocyte
growth. Namely, in situ hybridization studies (Bujo et
al., 1994) and electron microscopical immunocytochemistry (Shen et al., 1993) have shown that LR8- mRNA and protein are
present throughout the cytoplasm of previtellogenic oocytes. Upon onset
of oocyte growth, mRNA becomes localized to the periphery, and the
receptor is translocated to the plasma membrane (Shen et al.,
1993), where it initiates and maintains oocyte growth through yolk
precursor uptake. This translocation event could be a control site for
tight regulation of oocyte growth and may require oocyte-specific
mechanism(s) and protein factor(s). The absence of such mechanism or
its activation in COS-7 and Chinese hamster ovary cells would explain
the poor surface expression of LR8- in these cells. We also do
not know whether LR8- in muscle and heart (Fig. 4) do
reach the cell surface. LR8+ of somatic granulosa cells, which
surround the oocytes during all phases from quiescence to rapid growth,
may provide these cells with important substrates for their own
sustenance and/or production of oocyte-directed regulatory signals.
Current efforts are directed toward identification of these signals to
possibly delineate their precursors, among them physiological ligands
of LR8+. These considerations imply that ovarian LR8- and
LR8+ cooperate to regulate and support normal oocyte growth and
maturation. Detailed understanding of this synergism in the follicle
may identify principles and functions of LR8s that are equally
applicable to mammalian physiology.