(Received for publication, September 23, 1994; and in revised form, January 18, 1995)
From the
-Macroglobulin (
M), a major
plasma component in all vertebrates, is proposed to function as a broad
spectrum protease inhibitor. The
M-proteinase complex
(activated
M;
M
) is
removed rapidly by receptor-mediated endocytosis in the liver. Here we
demonstrate by Western blotting that
M is also present
in the yolk of chicken oocytes. Plasma levels of
M are
increased by estrogen, and yolk
M is partially
proteolyzed, consistent with the action of cathepsin D on endocytosed
M. Two known estrogen-induced ligands of the
oocyte-specific 95-kDa very low density lipoprotein/vitellogenin
receptor (OVR) are also fragmented by yolk cathepsin D (Retzek, H.,
Steyrer, E., Sanders, E. J., Nimpf, J., and Schneider, W. J.(1992) DNA Cell Biol. 11, 661-672). Since these findings
suggested a common uptake mechanism for lipoproteins and
M by oocytes, we investigated whether OVR, a member of
the low density lipoprotein receptor family, functions in the
metabolism of
M. Ligand blotting of oocyte membrane
extracts with chicken
M
revealed that it
binds to OVR. Surprisingly, the oocyte receptor also recognizes native
M, in sharp contrast to the hepatic receptor, which
only binds
M
. Receptor interaction of both
forms requires Ca
; however, competition experiments
suggest that
M and
M
interact with slightly different, or overlapping, sites on the
receptor. Colocalization of
M and OVR in coated
vesicles isolated from growing oocytes, and internalization and
degradation of methylamine-activated
M by COS-7 cells
transfected with OVR, strongly suggest that
M is
transported into growing oocytes via OVR. We propose that this
multifunctional receptor mediates pathways at the metabolic crossroads
of lipoproteins and protease inhibitor complexes.
During the last 7 days of the development of a hen's
oocyte, the giant cell takes up about 5 g of lipid and protein in the
form of very low density lipoprotein (VLDL) ()and
vitellogenin (VTG)(1, 2) . These major precursors of
yolk mass are synthesized in the liver and taken up by the oocytes from
the circulation via receptor-mediated endocytosis. Despite the absence
of obvious extensive structural similarity, VLDL and VTG are both
recognized by the same receptor, termed the oocyte VLDL/VTG receptor
(OVR), which is expressed at high levels in the plasma membrane of
growing female germ cells(3, 4) . The receptor was
purified from chicken follicles, and partial amino acid sequences
together with immunological evidence clearly showed that it belongs to
the LDL receptor gene family(5, 6) . Recent cloning of
OVR (7) revealed that it is the avian representative of the
so-called VLDL receptor family branch. The structural hallmark
characteristics of this
protein(8, 9, 10, 11, 12) were defined based on the structure of the rabbit
receptor(13) . The protein contains a cluster of eight
cysteine-rich complement-type binding repeats, in contrast to the seven
repeats found in all LDL receptors(14) . The term VLDL
receptor is based on the mammalian receptor's high affinity
for apolipoprotein (apo) E-containing lipoproteins, especially
VLDL(13) ; but in contrast to OVR, the true physiological
function(s) of mammalian VLDL receptors are not understood.
In this
respect it is of particular interest that the chicken OVR exerts a
broad ligand specificity, since it binds the major protein constituent
of VLDL, apoB(15) , VTG(3) , as well as mammalian apoE (16) , an apo not produced in
chicken(17, 18) . To date, an even wider ligand
specificity has been found for the LDL receptor-related
protein/-macroglobulin receptor
(LRP/
MR), another member of the LDL receptor gene
family(19) . This large membrane protein contains four clusters
of 2-11 binding repeats and can bind, at least in vitro,
such diverse ligands as
M(20) ; apoE (21) and apoE-enriched lipoproteins(22) ; lipoprotein
lipase(23) ; plasminogen activators and/or complexes with their
respective endogenous
inhibitors(24, 25, 26) ; receptor-associated
protein, a small 39-kDa intracellular protein that copurifies with the
receptor(27) ; lactoferrin(28) ; and rhinoviruses of
the minor group(29) . A homologue of this receptor is expressed
and has been characterized in somatic cells of the
chicken(30) . Besides functioning as a backup receptor system
for apoE-containing chylomicron remnants in
mammals(31, 32) , it serves as an
MR
in a wide variety of species (for review see (33) ).
The
LRP/MR rapidly removes from the circulation complexes
between proteinases and
M by specifically binding to a
receptor recognition site on
M. This recognition site
on
M is exposed by a conformational change, commonly
referred to as activation of
M (for review
see (34) ). The conformational change is the result of a
cleavage in the bait region of
M by a protease that
might become cross-linked to
M via an internal
thiolester of
M. Here we report that OVR, believed to
be the product of an ancestral gene involved in female reproduction,
binds not only activated
M, but also its native form.
Analysis of coated vesicles derived from small vitellogenic oocytes and
yolk from differently staged follicles suggests that
M
is a cytoplasmic constituent of chicken oocytes, which likely
endocytose it via OVR.
Electrophoretic transfer to nitrocellulose membranes (Hybond-C,
Amersham Corp.) was performed in 20 mM Tris-HCl, 0.15 M glycine buffer, pH 8.4, for 90 min at 200 mA. After transfer,
proteins were visualized by staining the membrane with Ponceau S (2
g/liter and 30 g/liter trichloroacetic acid) and rinsing with
HO. Nitrocellulose membranes used for Western blot analysis
were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20.
Bound polyclonal antibodies were detected with horseradish
peroxidase-conjugated protein A and an enhanced chemiluminescence (ECL)
system (Renaissance system, DuPont NEN). Membranes were exposed on
Reflection film (DuPont NEN) for the times indicated. Ligand blotting
with
I-
M was performed as described for
I-VLDL(6) , except that ligand-blotting buffer
contained 1 mM phenylmethylsulfonyl fluoride, which was added
immediately before use.
Figure 1:
Electrophoretic analysis of purified
chicken M.
M was purified from laying
hen plasma as described under ``Experimental Procedures'' and
analyzed by 4.5-18% SDS-gradient PAGE (panel A) and
nondenaturing PAGE (panel B). Panel A, 4 µg/lane
purified chicken
M (-, nonreducing; +,
reducing). Positions of migration of standard marker proteins are shown
on the left. Proteins were stained with Coomassie Brilliant
Blue. Panel B, nondenaturing PAGE of slow and fast forms of
purified human
M (lanes a-c) and
purified chicken
M (lanes d-f). Lanes a and d, native
M; lanes b and e,
MMA; and lanes c and f, trypsin-treated
M. 10 µg of
protein/lane was applied and stained with Coomassie Brilliant
Blue.
Figure 2:
Analysis of relative M
levels in serum samples. Panel A, 0.1 µl of the indicated
plasma samples was separated on a 4.5-18% SDS-gradient
polyacrylamide gel, and Western blotting was performed using
microimmunopurified anti-
M IgG as described under
``Experimental Procedures.'' Bands were visualized using the
ECL system according to the manufacturer's instructions; exposure
time was 3 min. IH, immature hen; LH, laying hen; IR, immature rooster; MR, mature rooster; ER, estrogenized rooster. Panel B, relative amounts
of
M in plasma were determined by densitometry of panel A, normalized to albumin levels as described under
``Experimental Procedures,'' and plotted in arbitrary units.
Designations are as in panel A.
Figure 3:
Ligand and Western blotting of oocyte
membrane proteins. Oocyte membrane Triton X-100 extracts (35 µg of
protein/lane) were subjected to electrophoresis on a 4.5-18%
SDS-gradient polyacrylamide gel, transferred to nitrocellulose, and
analyzed by ligand blotting (panel A) or a modified ligand
blotting procedure as described under ``Experimental
Procedures'' (panel B). Panel A, the blot was
incubated with I-labeled trypsin-treated chicken
M (
M
) (13 ng/ml, 56.800
cpm/ng) in the absence (lane 1) or presence (lane 2)
of 20 mM Na
EDTA. Lane 3 (no
Na
EDTA) and lane 4 (20 mM Na
EDTA) were incubated with
I-labeled
native chicken
M (26 ng/ml, 56,800 cpm/ng). The
autoradiograph was exposed for 44 h. Lane 5 is a Western blot
using rabbit anti-OVR IgG (16 µg/ml) and horseradish
peroxidase-protein A in conjunction with ECL. Exposure time was 2 min. Panel B, the blots were incubated with human trypsin-treated
M (6 µg/ml) in the absence (lane 1) or
presence (lane 2) of 20 mM Na
EDTA. Lane 3 (no Na
EDTA) and lane 4 (20 mM Na
EDTA) were incubated with native human
M (12 µg/ml). In lane 5, the ligand was
omitted as a control. Bound ligands were visualized with rabbit
anti-human
M IgG (30 µg/ml) as described under
``Experimental Procedures'' (lanes 1-5).
Exposure time 1 min. Lane 6 was obtained as described for lane 5 in panel A.
To determine if the binding of
native M to OVR was specific for the chicken ligand,
we performed ligand blots with purified human
M and
chicken oocyte membrane extracts. In this set of experiments (Fig. 3B), we used a sandwich ligand blot technique in
which bound ligands are visualized with specific antibodies against
human
M. This procedure eliminated the possibility
that
M became activated by the iodination process. As
with the chicken protein, not only trypsin-treated human
M (lane 1), but also the native form (lane 3) bound to the receptor in a
Ca
-dependent fashion (lanes 2 and 4). Importantly, when the procedure was performed without the
addition of activated or native
M, the antibody used
for the second step did not cross-react with any protein in the oocyte
membrane extract (lane 5). In Fig. 3B, lane 6, OVR is visualized with the specific antireceptor
antibody. The use of human
M also gave us the
opportunity to check electrophoretically for any ``fast''
form present in the preparation used in the ligand blotting
experiments. Even upon extreme overloading of the gel we could not
detect any fast form in freshly prepared human
M (cf. Fig. 1); thus, native
M from both chicken and
human serum bound to the chicken OVR.
When we tried to visualize the
binding of chicken M to the receptor by the same
procedure (i.e. without the labeling of chicken
M), the anti-chicken
M antibody
cross-reacted with too many bands present in oocyte membrane extracts,
making the results inconclusive.
The ability of chicken OVR to bind
both native and activated M prompted us to perform
experiments to determine whether the two forms and the major yolk
precursor ligands VLDL and VTG bound to common sites on the receptor.
In the first set of experiments (Fig. 4), we tested the ability
of activated and native
M to compete with the binding
of activated
M. As shown in Fig. 4A,
binding of activated
M can be competed for by both
native and activated
M. If native
M
was used as the labeled ligand (panel C), only the native form
effectively displaced the ligand from the receptor. Under these
experimental conditions, trypsin-treated
M, even at
almost 10,000-fold molar excess, had only little effect on the binding
of native
M (Fig. 4C, lane
5), suggesting that native and activated
M bound
to closely related or overlapping, but not identical, sites on the
chicken OVR.
Figure 4:
Cross-competition of M
and
M* with VLDL and VTG for the binding to OVR.
Oocyte membrane Triton X-100 extract (35 µg of protein/lane) was
subjected to electrophoresis on a 4.5-18% SDS-gradient
polyacrylamide gel under nonreducing conditions, transferred to
nitrocellulose, and ligand blotting was performed as described under
``Experimental Procedures.'' Panels A and B, the
blots were incubated with
I-
M
(7 ng/ml, 13,000 cpm/ng) with the following additions: panel
A: lane 1, none; lane 2, 3 µg/ml
M
; lane 3, 60 µg/ml
M
, lane 4, 3 µg/ml
M; lane 6, 60 µg/ml
M; panel B: lane 1, none; lane 2, 7 µg/ml
VTG; lane 3, 8 µg/ml VLDL; lane 4, 80 µg/ml
VLDL. Panels C and D, the blots were incubated with native
I-
M (7 ng/ml, 13,000 cpm/ng) with the
following additions: panel C: lane 1, none; lane
2, 3 µg/ml
M; lane 3, 60 µg/ml
M; lane 4, 3 µg/ml
M
; lane 6, 60 µg/ml
M
; panel D: lane 1,
none; lane 2, 7 µg/ml VTG; lane 3, 8 µg/ml
VLDL; lane 4, 80 µg/ml VLDL. Exposure times: panel
A, 6 h; panel B, 2 days; panel C, 6 h; panel
D, 2 days.
Next, we performed similar ligand binding competition
assays using as competitors the hitherto established ligands of the
oocyte receptor, VLDL and VTG(3) . Activated M
could be displaced only to a small extent by VTG and VLDL (Fig. 4B), whereas binding of native
M
appeared to be more sensitive to competition by VLDL (Fig. 4D); a 10,000-fold molar excess of VLDL abolished
the binding of native
M. VTG appeared similarly
effective, but we could not test its effect at the high concentrations
used for VLDL, since VTG tends to precipitate at conditions used for
ligand blotting (2 mM CaCl
; 37). These results
indicate that (i) the binding sites for native and activated
M on the
M receptor are closely
related to the binding site(s) for VLDL and VTG; and (ii) VLDL as a
competitor appears to discriminate between binding sites for native versus activated
M.
Figure 5:
Analysis of M in chicken
follicles. Panel A, Western blotting under nonreducing (lanes 1 and 3) and reducing (lanes 2 and 4) conditions of purified chicken
M (lanes 1 and 2; 45 µg/lane) and an enriched
fraction from yolk from vitellogenic follicles (lanes 3 and 4; 10 µg/lane) was performed using
microimmunoaffinity-purified rabbit anti-chicken
M IgG
(45 µg/ml) as described under ``Experimental
Procedures.'' Exposure time was 6 min. Panel B, analysis
of cathepsin D-digested purified chicken
M.
M (50 µg) was incubated with 3 units of cathepsin
D at pH 5.5 for 20 h at 37 °C (40) in the absence (lanes 5 and 6) or presence (lanes 7 and 8) of 1 µg of pepstatin A. Samples were analyzed by
SDS-PAGE under nonreducing (lanes 5 and 7) and
reducing conditions (lanes 6 and 8). The gel was
stained with Coomassie Brilliant Blue.
Figure 6:
Immunoblot analysis of M
in clathrin-coated vesicles from chicken oocytes. Proteins from coated
vesicles prepared from vitellogenic follicles (lanes 1 and 2, 50 µg/lane; lane 3, 20 µg) were separated
on a 4.5-12% SDS-gradient polyacrylamide gel under nonreducing (lanes 1 and 3) or reducing (lane 2)
conditions and transferred to nitrocellulose membranes. Lanes 1 and 2, Western blotting was performed with
microimmunoaffinity-purifed rabbit anti-chicken
M IgG
(45 µg/ml); exposure time was 6 min. Lane 3, Western
blotting was performed with rabbit anti-chicken OVR IgG (16 µg/ml);
exposure time was 30 s.
Figure 7:
Degradation of MMA by
COS-7 cells expressing OVR. COS-7 cells were transfected with a plasmid
carrying the full-length cDNA encoding for chicken OVR or the empty
plasmid as described under ``Experimental Procedures.'' On
day 2 after transfection, cell monolayers received 2 ml of standard
medium containing the indicated concentrations of
I-
MMA. After 5 h of incubation,
degradation products secreted into the medium were measured as amount
of trichloroacetic acid-soluble radioactivity recovered from the cell
supernatant. No-cell blanks were subtracted from the values obtained
for OVR-expressing and control cells. OVR-mediated degradation was
calculated by subtracting the values for control cells from those
obtained for OVR-expressing cells. Each value represents the average of
triplicate determinations.
There are several interesting aspects to the present finding
that OVR is positioned at the crossroads of lipoprotein and
M metabolism in the laying hen. First, we can gain
further insights into structure/function relationships of members of
the LDL receptor gene family. Recent cloning of chicken OVR (7) has revealed that it is an eight-binding repeat relative of
the LDL receptor and highly homologous to the mammalian VLDL
receptor(8, 9, 10, 11, 12, 13) .
Taking into account that OVR binds such structurally unrelated ligands
as VLDL and VTG(3) , apoE(16) , and
M,
as shown here, it presents itself as a multifunctional receptor like
its much larger relative, LRP/
MR.
LRP/
MR is a multiple-domain protein that contains 31
complement-type binding repeats clustered in four distinct subdomains
containing 2, 8, 10, and 11 of such repeats(19) , respectively.
As these clusters are separated from each other by long epidermal
growth factor precursor repeats, this large receptor can be envisioned
as a head-to-tail arrangement of functionally independent domains.
Possibly, such a cassette-like domain arrangement confers the observed
multiple ligand capacity to LRP/
MR. Recent attempts at
molecular dissection of the ligand binding sites on
LRP/
MR support this hypothesis, e.g. in that
activated
M and plasminogen activator/plasminogen
activator inhibitor-1 complexes bind to distinct regions of the
receptor protein(49, 50) . Considering the simple
structure of OVR (one set of eight repeats), structure/function
analysis of this multifunctional protein will facilitate greatly the
elucidation of minimal requirements for the binding of different
ligands.
Second, the physiological implications of the
identification of a second MR, which not only binds
activated
M like LRP/
MR, but also
interacts with native
M, are of interest. We are aware
that the experiments demonstrating the binding of native
M have to be evaluated carefully, since activation of
the native form, which might occur during isolation or labeling, could
lead to binding of the protein to the receptor. Importantly, analysis
on nondenaturing gels of freshly isolated chicken
M
did not detect the presence of any fast form of the protein (Fig. 1), which would have resulted from a bait region cleavage
by a protease. However, chicken
M treated with small
amines does not shift in electrophoretic mobility as human
M does, where such shift is interpreted as conversion
to an activated form. Thus, we also used as a ligand purified native
human
M, in which any activated form would have been
detected as the fast form. Significantly, preparations of native human
M, devoid of fast form when analyzed on nondenaturing
gel systems, strongly labeled the oocyte receptor in ligand blots. In
addition, we were able to use a sandwich ligand-Western blotting
procedure for studying the binding of human
M to OVR,
which avoids any alterations in the ligand possibly caused by labeling
procedures. This cross-species experiment demonstrates not only that
human
M can interact with an avian receptor but also
that the binding of native
M to OVR is not a
peculiarity of the chicken ligand, but is due to the properties of the
receptor.
Since being in the electrophoretically ``slow''
form may not suffice to indicate the presence of native
M, as suggested by van Leuven et
al.(51) , we also addressed the binding of both activated
and native forms of
M in competition ligand binding
studies. Experiments using activated and native
M,
VLDL, and VTG as unlabeled competitors demonstrated that binding of
native
M to OVR seems to be qualitatively different
from that of activated
M. In these in vitro experiments, activated
M does not compete
effectively with the binding of the native protein, and VLDL displaces
native
M completely, but activated
M
less efficiently. This finding is very similar to that described for
cross-competition of VTG and VLDL to OVR (5, 52) . In
that case, unlabeled VTG competed for the binding of labeled VTG and
VLDL, whereas VLDL did not effectively block binding of VTG to OVR. In
analogy to VLDL/VTG, we interpret the current findings to indicate that
the binding site for activated
M represents a
substructure of the recognition site for native
M. It
is conceivable that the presence of native
M in the
binding site decreases the affinity for activated
M,
but the reverse appears not to be the case. Taken together, these
results demonstrate for the first time the existence of a receptor for
activated and native
M.
In addition to finding an
M receptor in the oocyte membrane, we could
demonstrate that
M is a component of chicken yolk. Its
presence and colocalization with the receptor in clathrin-coated
vesicles derived from chicken follicles are consistent with
receptor-mediated transport of
M from the plasma
compartment into growing oocytes. This notion is supported by two
additional findings. First, plasma concentrations of
M
are significantly increased upon estrogen administration, which is an
important common property of serum-borne yolk precursors like VLDL,
VTG, and riboflavin-binding protein(53) . Second, we used
transformed COS-7 cells expressing OVR to demonstrate directly that
M undergoes OVR-mediated endocytosis. For two reasons,
these experiments were performed with
MMA only. First,
trypsin-activated
M, even after careful gel
chromatographic removal of excess trypsin, resulted in partial
detachment of the cells from the culture dishes. Second, since native
M becomes activated under incubation conditions (37
°C, 5 h) used for the degradation experiments, data obtained would
not reflect uptake of native
M.
Nevertheless, COS-7
cells expressing OVR specifically take up and degrade
MMA, demonstrating endocytotic competence of OVR
toward at least one conformational form of
M. In this
respect, a recently published result by Andreasen et al.(54) needs consideration. There, the purification of a
VLDL receptor from bovine mammary gland was reported, and the same
protein was demonstrated to be expressed in a human mammary carcinoma
cell line. Using these cells, the authors could not show binding to and
degradation of
M via this protein. This contradictory
finding could have several causes. First, the identity of this protein
as the VLDL receptor was suggested based on amino-terminal protein
sequencing only, leaving the possibility that it is a homologous
protein, but in fact not the VLDL receptor. Second, binding of
M could be restricted to the chicken representative of
the VLDL receptor group, which is the key player in oocyte growth via
receptor-mediated endocytosis of several yolk precursors. However, this
explanation is rendered less likely by the very high degree of
similarity of the primary structures of OVR and mammalian VLDL
receptors. Third, the endogenous expression of the VLDL receptor in
mammalian tissues and cultured cells appears to be extremely low. This
is the major obstacle to establishing unequivocally the ligand binding
specificity of this receptor in mammalian systems. The problem is
confounded by the fact that most potential ligands also bind to
LRP/
MR, which is expressed abundantly in these cells.
As shown here for
M, chicken OVR, expressed at very
high levels in follicles, has already demonstrated its advantages as a
superior system to study physiological ligands of this new group of LDL
receptor-related proteins.
At present, we do not know the exact
conformational species of M which binds to the
receptor in vivo and is transported into the oocyte. We have,
however, obtained initial insights into this aspect by analysis of the
content of clathrin-coated vesicles, which represent the earliest
isolatable structures of the endocytic pathway. Coated vesicles from
small vitellogenic follicles (5-6 mm in diameter) (4) contain a mixture of the 180-kDa form and the 85-kDa
fragment of
M. Since
M is a
homotetrameric protein, this result could indicate either the presence
of a mixture of bait region cleaved and native
M or of
partially proteolyzed
M. In an attempt to resolve this
question, we isolated coated vesicles from previtellogenic follicles
(1-2 mm in diameter), whose oocytes had not yet entered the rapid
growth phase. Despite the low yield of vesicles from these follicles,
we found that most of the immunoreactive material migrated as the
180-kDa subunit and that only traces of the 85-kDa fragment of
M were present (data not shown). Since there is no
indication that coated vesicles derived from previtellogenic or
vitellogenic follicles are functionally or structurally different, we
suggest that the cleaved form seen in preparations derived from
vitellogenic follicles is most likely a contamination from bona fide
yolk, i.e. endosomal yolk spheres (4 and references therein).
Yolk spheres comprise the final, membrane-bound compartment for storage
of endocytosed yolk proteins and make up the body of yolk (4) .
Being the end product of vesicular endocytic activity, they contain the
proteolytically processed form of yolk components, e.g. the
cleaved form of
M. In turn, the previtellogenic
follicles we analyzed do not contain significant amounts of yolk, and
vesicles prepared thereof are less likely contaminated with yolk
spheres. In experiments not shown, we repeatedly washed the vesicle
preparation from vitellogenic follicles and observed continuous loss of
only the 85-kDa band.
Based on the properties of OVR, it is
intriguing to speculate that growing oocytes preferentially take up
native M, i.e. the prevalent form of this
protein in plasma, consistent with a continuous flow from the plasma
compartment into rapidly growing oocytes. Significant oocyte uptake of
activated
M would imply a mechanism that would
locally, probably in the ovarian tissue, or systemically produce
activated
M in continuous fashion. To us it seems
unlikely that continuous protease activation of
M, a
process underlying its proposed primary function in vertebrates, should
be necessary for the transport of a yolk component into the oocyte. As
a corollary, the oocyte, because of its abundance of OVR(5) ,
and not the liver, in which OVR is not expressed(7) , would
serve as the major sink of functionally expended
M,
which is equally unlikely. However, we cannot rule out that there
exists a specific mechanism in the chicken to produce
transport-competent
M, different from the activation
by the reaction with proteases.
We envision two possible functions
for M in the yolk. First, since partial proteolytic
digestion of yolk precursors appears to be a prerequisite for their
deposition in yolk,
M may serve subsequent to
endocytosis to inactivate cathepsin D within the yolk and during early
embryogenesis. In this case, cathepsin D must be kept active prior to
its reaction with other yolk precursors. Second, considering the
efficacy of the uptake process to sustain the reproductive effort of
the hen (12-15 g of yolk are produced every 25 h), the transport
of
M into the oocyte may serve as piggyback mechanism
for a variety of other substances. Recently, we have described the
receptor-mediated cotransport of riboflavin-binding protein and
VTG(53) . Candidate molecules for transport via
M are growth factors and other small cytokines, many
of which have been shown to have high affinity for
M
(for review see (55) ). Such a transport system involving OVR,
a multifunctional receptor capable of binding different ligands that
can serve as carriers for other minor yolk precursors, seems to be an
elegant and efficient way to meet the requirements of the rapidly
growing female germ cell.
The fact that chicken OVR is highly
homologous to mammalian VLDL receptors together with our finding that
it also binds M have implications for the evaluation
of the function(s) of this receptor in mammals, where this is still a
matter of debate. The intriguingly high conservation of this protein in
egg-laying species and mammals does point to a very important common
function. In this respect, the recent discovery that significant levels
of the VLDL receptor mRNA are present in placenta of man(12) ,
mouse (11) , and rat (56) might indicate that the
mammalian VLDL receptor is part of a multifunctional transport
machinery in the very tissue that continuously supplies the growing
embryo with nutrients, vitamins, growth factors, and possibly other
components. Therefore, the VLDL receptor could play a key role in
mammalian reproduction similar to that demonstrated here for OVR in a
nonplacental species.