(Received for publication, August 14, 1995; and in revised form, March 5, 1996)
From the
Transcobalamin II receptor (TC II-R) exists as a monomer and a
dimer of molecular masses of 62 and 124 kDa in the microsomal and
plasma membranes, respectively, and in vitro, pure TC II-R
monomer dimerizes upon insertion into egg PC/cholesterol (molar ratio,
4:1) liposomes (Bose, S., Seetharam, S., and Seetharam, B.(1995) J.
Biol. Chem. 270, 8152-8157 and Bose, S., Seetharam, S.,
Hammond, T., and Seetharam, B.(1995) Biochem. J. 310,
923-929). The current studies were carried out to define the
mechanism of TC II-R dimerization. Both the mature TC II-R (62 kDa) and
the enzymatically deglycosylated TC II-R (45-47 kDa) demonstrated
optimal association and formed dimers of molecular masses of 95 and 124
kDa, respectively, at 22 °C when bound to egg PC vesicles
containing at least 10 mol % of cholesterol. Mature TC II-R dimerized
upon insertion into synthetic phosphatidylcholine vesicles of different
fatty acyl chain length (dimyristoyl, dipalmitoyl, and disteroyl
phosphatidylcholine) in the absence or the presence of cholesterol at
temperatures below or above their transition temperatures,
respectively. Dimerization of TC II-R also occurred with vesicles
prepared using lipid extract from the plasma but not microsomal
membranes. Cholesterol depletion of native intestinal plasma membranes
or its enrichment in the microsomal membranes resulted in the in
situ conversion of the 124-kDa dimer to the 62-kDa monomer or of
the monomer into the dimer form, respectively. Treatment of plasma
membranes with phospholipase A resulted in the conversion
of the dimer form of the receptor to the monomer form and spin label
studies using 1-palmitoyl, 12 doxylsteroyl phosphatidylcholine revealed
that interactions of TC II-R with PC vesicles increased order around
the probe. Based on these results we suggest that dimerization of TC
II-R is mediated by its interactions with a rigid more ordered lipid
bilayer membrane, is regulated in plasma membranes by cholesterol
levels, and is independent of glycosylation-mediated folding.
Plasma transport of absorbed Cobalamin (Cbl; vitamin
B) (
)to tissues is mediated by transcobalamin
II (TC II), a 43-45-kDa nonglycoprotein plasma Cbl
binder(1) . The tissue/cellular uptake of TC II-Cbl occurs via
receptor-mediated endocytosis (2) and is mediated via TC II-R
expressed in the plasma membranes of all
tissues(3, 4, 5) . The importance of plasma
membrane expression of TC II-R in plasma transport of Cbl is borne out
by our recent observation (6) that in vivo, functional
inactivation of TC II-R by its circulating antibody results in the
development of Cbl deficiency. Recent studies (4) from our
laboratory have shown that TC II-R exists as a dimer of molecular mass
of 124 kDa in all tissue membranes and that the dimerization of TC II-R
is due to noncovalent interaction between two monomers of molecular
mass of 62 kDa and in vitro is mediated by its interactions
with lipid bilayer prepared using egg PC and cholesterol. Our recent
immunoblot studies (4, 5, 6) have identified
TC II-R dimers in the tissue membranes across species, and in the rat
kidney, the 62 kDa monomer was present only in the microsomes, whereas
the 124-kDa dimer form of the receptor was the only form of TC II-R
present in the apical and basolateral membranes, intermicrovillar
clefts, and clathrin-coated vesicles. Neither the monomer nor the dimer
form of the receptor was detected in the light endosomes and
lysosomes(5) . The steady state level of TC II-R dimer is
8-10-fold higher than that of TC II-R monomer in all the tissue
membranes tested(5) . Taken together, these studies (4, 5) have indicated that dimerization of TC II-R is
a rapid, post-microsomal event and that the inability of the TC II-R to
dimerize in the microsomal membranes could be due to (a) lack
of lipid microenvironment essential for its dimerization, (b)
improper folding of TC II-R due to incomplete maturation of its N- and/or O-linked sugars, or (c) weaker
interaction between the two monomers such that the treatment with SDS
results in the disassociation of the dimer.
The present investigation was carried out to further explore the mechanism of TC II-R dimerization and to examine which of the abovementioned possibilities are operational in mediating the noncovalent interaction between the two monomers of TC II-R. The results of the current study show that (a) in vitro, the dimerization of TC II-R is regulated by a rigid, more ordered lipid bilayer, (b) in vivo, higher cholesterol levels of plasma membranes provide a more ordered lipid microenvironment to facilitate the dimerization of TC II-R, and (c) the dimerization of TC II-R is not influenced by folding alterations due to its N- and O-glycosylation.
The following chemicals were purchased as indicated: egg PC,
dimyristoyl PC, dipalmitoyl PC, disteroyl PC, 1-palmitoyl
12-doxylsteroyl-sn-glycero-3-phosphocholine, and cholesterol
(Avanti Polar Lipids Inc. Alabaster, AL); [Co]
cyanocobalamin (15 µCi/µg) and carrier-free Na
I (Amersham Corp.); peptide N-glycosidase from Flavobacterium menengosepticum and O-glycosidase from Diplococcus pneumoniae (Boehringer Mannheim); phospholipase
A
from bee venom, phospholipase C from Clostridium
Welchi, phospholipase D from peanut, sialidase from Clostridium Perifringens, dihydrocholesterol, 7-keto
cholesterol, and protein A (Sigma).
Pure TC II-R was obtained from
human placenta essentially as described earlier(4) .
Monospecific antiserum to TC II-R was prepared in rabbits as described
earlier(4) . The ligand TC II used in the TC II-R assays was
partially purified from human plasma according to Lindemans et
al.(7) . TC II-R activity in the lipid vesicles was
determined using human TC II-[Co]Cbl (2 pmol)
and the Triton X-100 extracts of the reconstituted vesicles by the
DEAE-Sephadex method of Seligman and Allen(8) .
Cholesterol depletion in the basolateral membranes were carried out as follows. Basolateral membrane (2.5 mg of protein) in 0.5 ml of 10 mM Tris-HCl buffer containing 140 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride was treated with 5 µg of digitonin for 15 min at 22 °C. The membrane collected by centrifugation was washed three times with 2 ml of Tris-buffered saline and resuspended in 500 µl of the same buffer. Cholesterol and phospholipid levels were estimated in the lipid extract of digitonin treated membranes as described above.
Cholesterol enrichment of the microsomal membranes was carried out as follows. Cholesterol (25 µg) in chloroform was dried under nitrogen. Microsomal membrane (1 mg of protein) in 1 ml of Tris-HCl buffer was added, vortexed, and incubated for 1 h at either 37 or 5 °C. The membrane thus treated was collected by centrifugation, washed, and resuspended in the same buffer. In some experiments, cholesterol was replaced by its analogues, dihydrocholesterol, and 7-ketocholesterol. Phospholipid and cholesterol (or sterol) levels in these membranes were determined as before.
Phospholipase digestion of basolateral membranes was carried out as
follows. Basolateral membranes (300 µg) in 50 µl were digested
with phospholipase A (15 units, 30 min), phospholipase C
(7.5 units, 240 min), or phospholipase D (13 units, 30 min).
Figure 1:
Optimization of transfer time during
immunoblotting of TC II-R monomer and dimer forms. A, rat
intestinal mucosal total membranes (100 µg) were separated on
SDS-PAGE (7.5%). Each lane containing the separated proteins was
transferred onto nitrocellulose for the indicated time intervals. The
transferred proteins were probed with antiserum to human TC II-R and I-protein A. The top strip represents the
124-kDa dimer form, and the bottom strip represents the 62-kDa
monomer form. The time of exposure to detect both the dimer and monomer
forms was the same (18 h). B, rat intestinal total membranes
(300 µg) were separated on SDS-PAGE (7.5%). The separated proteins
were transferred to nitrocellulose for 60 min and probed as before. The
time of exposure was 80 h. Both SDS-PAGE and the immunoblotting were
carried out in triplicate, and the same results were obtained each
time.
Figure 2:
TC
II-R association with egg PC vesicles containing increasing mol % of
cholesterol. Pure placental TC II-R (1 µg) was added during the
preparation of egg PC vesicles. Cholesterol levels in these vesicles
were increased from 0-50 mol %, and the concentration of egg PC
used was 2 µmol. Following dialysis the liposomally bound TC II-R
was collected by centrifuging at 150,000 g for 2 h.
The supernatant (
) and the lipid pellet (
) were assayed (a) for TC II-[
Co]Cbl binding. The
liposomal pellet was separated on SDS-PAGE and immunoblotted, and the
bands were quantified by AMBIS radioimaging system (b). One
unit of TC II-R associated is equal to 20,000 counts of image density.
The reconstitution experiment using various concentration of
cholesterol was carried out in triplicates. The results shown represent
the means ± S.D. from duplicate assays from each of these
reconstitution experiments.
Upon SDS-PAGE and
immunoblotting of the liposomally bound TC II-R (Fig. 3), the
monomer form of 62 kDa was associated with the liposomes prepared with
using 1 (Fig. 3a, lane 2) or 2 mol % (Fig. 3a, lane 1) of cholesterol (Fig. 3a) but not with the liposomes prepared using
>10 mol % of cholesterol (Fig. 3a, lanes
3-5). On the other hand, the 124-kDa dimer was present only
when the cholesterol content of the egg PC liposomes was 10 mol % (Fig. 3b, lanes 3-5) but not when the
mol % of cholesterol was 1 or 2 (Fig. 3b, lanes 1 and 2). Quantitation of the immunoblots revealed that TC
II-R protein association (Fig. 2b) increased by about
9-fold with an increase in cholesterol content of the liposomes from 1
to 50 mol %. These results suggested that the association of TC II-R
with egg PC bilayers is dependent on the presence of cholesterol and
that its optimal binding and dimerization occurred when the mol % of
cholesterol was at least 10. In order to further examine the role of
cholesterol in the lipid bilayer and its effect on the dimerization of
TC II-R, reconstitution experiments were carried out using synthetic
symmetrical PCs of different length fatty acyl residues in the presence
and the absence of cholesterol and at temperatures above and below
their phase transition.
Figure 3:
Immunoblot analysis of liposomally bound
TC II-R. The liposomal pellet was subjected to SDS-PAGE (7.5%),
transferred to nitrocellulose for 45 (a) or 90 min (b), and probed with diluted antiserum to placental TC II-R
and I-protein A. a, lanes 1-5 represent TC II-R associated with egg PC vesicles containing 2, 1,
10, 20, and 50 mol % of cholesterol. b, lanes 1-5 correspond to 1, 2, 10, 20, and 50 mol % of
cholesterol.
Figure 4: Immunoblot analysis of TC II-R associated with DMPC, DPPC, and DSPC vesicles. a, TC II-R bound to DMPC (lanes 1 and 4), DPPC (lanes 2 and 5), and DSPC (lanes 3 and 6) vesicles below their respective transition phase temperatures in the absence (lanes 1, 2, and 3) and the presence (lanes 4, 5, and 6) of 50 mol % cholesterol were analyzed on SDS-PAGE (7.5%) and subjected to immunoblot analysis as before. Liposomal preparation were carried out at either 5 °C for DMPC vesicles or room temperature for DPPC and DSPC vesicles. b, TC II-R bound to DMPC (lanes 1 and 4), DPPC (lanes 2 and 5), and DSPC (lanes 3 and 6) vesicles above their respective phase transition temperatures in the presence (lanes 1, 2, and 3) and the absence (lanes 4, 5, and 6) of 50 mol % cholesterol were analyzed on SDS-PAGE and subjected to immunoblot analysis. Liposomal preparation was carried out at 37, 45, and 65 °C using DMPC, DPPC, and DSPC, respectively.
Figure 5: Immunoblot analysis of TC II-R bound to liposomes prepared using microsomal and plasma membrane lipids. Pure TC II-R (25 ng) bound to liposomes prepared using lipid extracts from microsomal (lane 1 and 5), basolateral (lanes 3 and 7) membrane lipids, native microsomal membrane (50 µg of protein, lanes 2 and 6), and basolateral membranes (5 µg of protein, lanes 4 and 8) were subjected to SDS-PAGE and immunoblotted as before.
Figure 6: Immunoblot analysis of TC II-R monomer and dimer in cholesterol depleted basolateral (top panel) and cholesterol enriched microsomal (bottom panel) membranes. Top panel, immunoblot analysis of BLM (5 µg of protein) untreated (lanes 1 and 6), digitonin-treated (lanes 2 and 7), or Triton X-100-treated (lanes 4 and 9) or untreated microsomal membranes (50 µg of protein, lanes 3 and 8) or Triton X-100-treated microsomes (lanes 5 and 10). Bottom panel, immunoblot analysis of microsomal membranes, untreated (lanes 1 and 6), treated with cholesterol at 5 °C (lanes 2 and 7) or treated at 37 °C with dihydrocholesterol (lanes 3 and 8), 7-ketocholesterol (lanes 4 and 9), and cholesterol (lanes 5 and 10). The transfer time during immunoblotting in both top and bottom panels was either 45 (a) or 90 min (b).
In contrast to the in situ conversion of the TC II-R dimer to the monomer form upon cholesterol depletion of the basolateral membranes, immunoblot analysis (Fig. 6, bottom panel) of the microsomal membranes enriched in cholesterol resulted in the conversion of the monomer form (Fig. 6a, lane 1) to the dimer form (Fig. 6b, lane 10). The conversion in the physical state of TC II-R from the monomer to the dimer form occurred only when the microsomal membranes were incubated with cholesterol at 37 °C (Fig. 6b, lane 10) but not at 5 °C (Fig. 6b, lane 7). The specificity of cholesterol effect on the in situ conversion of the TC II-R monomer to the dimer form in the microsomal membrane is borne out by the observation that treatment of microsomal membranes with cholesterol analogues such as either dihydrocholesterol (Fig. 6, a, lane 3, and b, lane 8) or 7-ketocholesterol (Fig. 6, a, lane 4, and b, lane 9) had no effect on the physical state of TC II-R. Following treatment of the microsomal membranes with these analogues, TC II-R remained as a monomer (Fig. 6a, lanes 3 and 4) without conversion to the dimer form (Fig. 6b, lanes 8 and 9). However, the treatment of microsomal membranes altered the phospholipid to cholesterol (or sterol) ratio from 26:1 in the native membranes to 18:1, 10.2:1, 11.33:1 and 9.8:1 following treatment of microsomal membranes with cholesterol at 5 °C or at 37 °C or with dihydrocholesterol or 7-ketocholesterol at 37 °C, respectively. Taken together, the results from both the native membranes and synthetic PC bilayers indicated that the association and dimerization of TC II-R may be due to phospholipid-cholesterol interactions, a factor that influences the fluidity (order) of these membranes.
Figure 7:
Effect of phospholipase A, C,
and D treatment on the molecular mass of basolateral membrane TC II-R.
Untreated BLM (10 µg) (lanes 1 and 5) or digested
with phospholipase C (lanes 2 and 6), phospholipase D (lanes 3 and 7), or phospholipase A
(lanes 4 and 8) were subjected to SDS-PAGE
(7.5%), and the separated proteins blotted onto nitrocellulose for 90 (a) or 45 min (b) were probed with diluted antiserum
to human TC II-R and
I-protein A. The bands were
visualized by autoradiography.
Figure 8:
EPR spectra of 12PCSL in liposomes
prepared from basolateral membrane lipids (A and B)
or microsomal membrane lipids (C and D). Samples B and D contained TC II-R (lipid/protein,3000:1),
while samples A and C contained lipid only. The spin
label concentration was 1 mol % relative to total lipids. Spectra were
obtained at room temperature (22 °C) and are the average of eight
scans. The total scan width was 100 Gauss. Inset beneath each spectrum
is a 5 amplification of the high field region emphasizing the
strongly immobilized component (arrow in B) observed
only in membranes containing dimeric TC
II-R.
EPR spectra of the phospholipid spin label, 12PCSL, in liposomes prepared from either basolateral or microsomal lipids with and without TC II-R are shown in Fig. 8. Motion of the spin label was significantly restricted in basolateral liposomes containing TC II-R (Fig. 8B) relative to the other three systems, as indicated by the increased line widths and appearance of a strongly immobilized high field component in the EPR spectrum. Given the low protein content (protein/lipid, 1:3000) in the reconstituted system, this strongly suggests direct interaction of the dimeric receptor with the sn-2-acyl chain of PC that bears the nitroxide label. No such interaction was observed in TC II-R-containing liposomes composed of microsomal lipids, where the receptor is monomeric (Fig. 8D). The immobilization was not due to differences in the lipid phase alone, because the rotational motion of the spin label remained fast in both basolateral and microsomal lipid membranes lacking TC II-R (Fig. 8, A and C). Similar effects were observed in purely model systems and correlated well with dimer formation as observed by SDS-PAGE (e.g., Fig. 4and 5), where the EPR spectrum of 12PCSL in DMPC/cholesterol liposomes (4:1) at 37 °C, containing TC II-R was significantly broadened relative to that in DMPC/cholesterol (4:1) alone at 37 °C or to that of DMPC vesicles prepared at 37 °C with or without TC II-R (at 37 °C) (data not shown).
Figure 9: SDS-PAGE analysis of enzymatically deglycosylated TC II-R bound to egg PC/cholesterol liposomes. Egg PC/cholesterol vesicle bound untreated (lane 1), N-glycanase-treated (lane 2), sialidase and O-glycanase treated (lane 3), and N-glycanase, sialidase, and O-glycanase treated (lane 4) TC II-R (2 µg) was subjected to SDS-PAGE (7.5%), and the protein bands were visualized following staining of the gel with silver nitrate.
In previous studies(4, 5, 6) , an immunoblotting procedure to detect and quantify the monomer and the dimer forms of TC II-R was used. This method used a transfer time of 45 min to detect the monomer and 90 min to detect the dimer, respectively. In the current studies, the time of transfer used for the optimal transfer of the monomer and the dimer form has been validated (Fig. 1A). The data shown in Fig. 1A when quantified demonstrated a monomer to dimer ratio of 1:8, a value similar to that obtained earlier for the distribution of TC II-R monomer and dimer forms in several rat tissue total membranes (5) . A single transfer time of 60 min (Fig. 1B) can be used to detect both the species of TC II-R in a single gel. However, it will not accurately reflect the absolute steady state amounts of the two species present in any given membrane or the interconversion of one form to the other during experimental modulation of the membranes. When the data shown in Fig. 1B was quantified, the ratio of monomer to dimer was 1:2. This value is different than the value of 1:8 obtained using two different transfer times, 45 min to transfer the monomer and 90 min to transfer the dimer. The lower ratio reflects only partial transfer of the dimer and a partial loss of the monomer during the 60-min transfer of the protein from the gel. The decreased transfer of the dimer could be visualized during the 60-min transfer of pure TC II-R bound to PC/cholesterol vesicles by staining the gel for protein. The loss of TC II-R monomer during the 60-min transfer could also be visualized when pure TC II-R was immunoblotted onto a second nitrocellulose filter (data not shown). The likely explanation for the 2-fold time differential for the optimal transfer from SDS-PAGE of the two forms of TC II-R to the nitrocellulose membranes could be that this phenomena is related to the receptor status, i.e., a monomer or a dimer.
In the present work we have addressed issues related to the mechanism of TC II-R dimerization in phospholipid bilayers and in native membranes. Previously (4) we have shown that in native plasma membranes, TC II-R that exists as a dimer of molecular mass of 124 kDa could be converted to a 62-kDa monomer with solubilization of the receptor by Triton X-100 or without its solubilization by delipidation of the plasma membranes with 2:1 mixture of chloroform and methanol. These results clearly indicated that the dimerization of TC II-R is mediated by its strong interactions with the membrane lipids. Some insight into nature of the membrane that may mediate the dimerization of TC II-R was obtained from further studies (5) aimed at understanding the intracellular distribution of TC II-R in the rat kidney. These studies (5) demonstrated that under identical conditions of immunoblotting (except the transfer time), the monomer form of TC II-R with a molecular mass of 62 kDa was detected only in the microsomal membranes, whereas the dimer form of TC II-R with a molecular mass of 124 kDa was the only species present in the plasma membranes. In addition, these studies showed that in many rat tissues, the dimer form of TC II-R was the predominant form present.
One major difference between the plasma and microsomal
membranes of many mammalian tissues is the molar ratio of phospholipid
to cholesterol. In the plasma membranes the ratio is about
1-1.2:1(14, 15) , whereas it is about 22:1,
20:1, and 26:1 in the microsomal membranes isolated from rat kidney,
placenta, and intestinal mucosa, respectively. ()Because TC
II-R exists in two different physical states in these two membranes, we
hypothesized that TC II-R dimerization in the native plasma membranes
may be related to the presence of higher amount of cholesterol in these
membranes. This hypothesis was further based on our earlier observation
that TC II-R is able to dimerize with egg PC vesicles that contained 25
mol % of cholesterol(4) . In order to explore this hypothesis,
additional studies were carried out using both phospholipid vesicles
and native microsomal and plasma membranes isolated from rat intestinal
mucosa.
Based on the experimental evidence from the current study, we conclude that the single most important factor affecting the dimerization of TC II-R in native plasma membranes and lack of dimerization in the microsomal membranes is the difference in their relative cholesterol content. This conclusion is based on the three lines of evidence. First, the association (Fig. 2) and the ensuing dimerization of TC II-R with egg PC occurred with a minimum cholesterol content of 10 mol % (Fig. 2). At cholesterol levels of 1 or 2 mol %, TC II-R associated poorly with egg PC vesicles and stayed as a monomer. Second, mature TC II-R dimerized upon insertion into lipid vesicles prepared using total lipids from intestinal basolateral but not microsomal membranes (Fig. 5). The phospholipid to cholesterol ratios in these extracts were 1.2:1 and 26:1, respectively (data not shown). Third, changing the phospholipid/cholesterol ratio from 1.2:1 to 26:1 in the basolateral plasma membranes by cholesterol depletion with digitonin treatment (Fig. 6, top panel) or from 26:1 to 10:1 in the microsomal membranes by cholesterol enrichment (Fig. 6, bottom panel) resulted in the in situ conversion of the dimer form to the monomer form and from the monomer form to the dimer form, respectively.
How does cholesterol influence the
interaction between the two monomers of TC II-R in order to facilitate
the formation of noncovalent dimers in the native membranes? The answer
may be related to the potential function(s) of cholesterol in
membranes. Cholesterol could play a role in facilitating the
hydrophobic match between the lipid bilayer and TC II-R, thus leading
to its association and dimerization. Hydrophobic matching is a well
established model for lipid-protein interactions in the
membranes(16, 17, 18) . This model states
that in order to accommodate or match the hydrophobic region of a
protein with that of the hydrophobic core of lipid bilayer, association
of a protein is accompanied by alterations in the thickness of lipid
bilayers. Earlier studies (19, 20, 21) have
confirmed that physiological membrane functions are controlled by
hydrophobic membrane thickness. For example, optimal activity of
Ca-ATPase (22, 23) and acetylcholine
receptor (24, 25) function can be altered by the
introduction of n-alkanes, which are known to increase
hydrophobic thickness of the lipid bilayer. Like n-alkanes,
cholesterol is also known to increase the hydrophobic thickness of
lipid bilayers(26, 27) . Therefore one possible
explanation for the cholesterol induced effects of TC II-R association
and the ensuing dimerization could be that it is due to the
cholesterol-mediated thickening of lipid bilayer. During such a
thickening, hydrophobic matching between TC II-R and the hydrophobic
core of the membrane could facilitate two monomers of TC II-R to be
spatially near to one another and dimerize encompassing a specific
lipid milieu within the bilayer.
Based on experimental evidence, a
more likely possible role of cholesterol in the dimerization of TC II-R
is that it is due to interaction of cholesterol with the phospholipid.
X-ray and neutron scattering studies have shown that cholesterol
inserts normal to the plane of the bilayer with the -OH group near
the ester carbonyl of the lipid(28, 29) . It is
thought that this results in the development of hydrogen bonding
between the
-hydroxyl group of cholesterol and the carbonyl oxygen
linking the fatty acyl chains with its glycerol backbone(30) .
However, Raman spectroscopy indicates that no actual hydrogen bonding
occurs with these carbonyls(31) . Despite this uncertainty,
what is generally agreed upon is that cholesterol has a substantial
effect on the order parameters measured along the lipid hydrocarbon
chain by
H-NMR (32) and on the phase transition of
the phospholipid(33) . Fourier transform infrared spectroscopy
studies (34) have shown that above the transition midpoint
cholesterol decreases the fraction of gauche rotomers in the
phospholipid hydrocarbon chain, whereas just the opposite is the effect
below the transition midpoint.
Effect of cholesterol on the membrane
interactions of TC II-R are mediated by increasing the order around the
fatty acyl residues. Several lines of evidence support that the order
around the 2-fatty acyl residue is important for the interactions of TC
II-R with lipid bilayers. One, cholesterol analogues were unable to
mediate the dimerization of TC II-R in the fluid microsomal membrane (Fig. 6), suggesting very strongly that the cholesterol-induced
dimerization of TC II-R is due to increased interaction of the highly
hydrophobic TC II-R with a rigid microdomain of the bilayer. Thus, with
only 4% cholesterol, a more fluid microsomal membrane will not support
TC II-R dimerization, as opposed to the basolateral membrane, a more
ordered (less fluid) membrane with nearly 40% cholesterol. Two, the
ability of phospholipase A but not other phospholipases to
convert in situ, the TC II-R dimer to the monomer form and the
ESR studies support the concept that TC II-R dimerization is due to
phospholipid fatty acyl chain-cholesterol interaction. Three, the role
of cholesterol in increasing the rigidity of phospholipid bilayers
above their transition temperature is well
accepted(35, 36, 37) . The results obtained
with DMPC, DPPC, and DSPC show that cholesterol-induced dimerization
occurred above but not below the transition temperature of these PCs,
once again suggesting that increased association and the ensuing
dimerization of TC II-R is due to cholesterol-mediated increase of
bilayer rigidity/molecular order or decreased fluidity. It is
interesting to note that the addition of 50 mol % cholesterol to these
vesicles below their transition temperature resulted in very poor or no
binding of TC II-R, clearly indicating that disappearance of phase
transition (38) is not conducive for the association and
dimerization of TC II-R.
One of the important regulators of
intracellular folding of proteins is its post-translational
modifications. With respect to TC II-R, these include maturation of a
single N-linked oligosaccharide to the complex type and the
maturation of its O-linked sugars. TC II-R contains several O-linked sugar residues, and the contribution of its molecular
mass of 62 kDa by the O-linked sugars is about 15 kDa and by N-linked sugars is about 2 kDa. Due to the presence of at
least 27-28% of sugars in TC II-R, it is likely that they may
play a role in influencing the folding of TC II-R required for its
interactions with the lipid bilayer and the ensuing dimerization. The
results (Fig. 9) convincingly show that the dimerization of TC
II-R is independent of the potential folding alterations of the
receptor during its carbohydrate maturation and intracellular
trafficking. Other potential co- and post-translational modifications
of TC II-R that could play a role in its interactions with the lipid
bilayer and influence its dimerization include different types of
covalent lipid modification. TC II-R expressed in either
intestinal-derived Caco-2 cells or proximal tubular-derived opossum
kidney cells when labeled with [H]myristate or
palmitate or mevelonate failed to incorporate the label into TC II-R,
although other proteins expressed in these cell lines were labeled with
one or the other of these labels. In addition, incubation of Caco-2
cells with cerulenin, an inhibitor of palmitoylation of proteins (39) failed to inhibit either the surface domain expression or
the T
of TC II-R, suggesting that TC II-R is not
palmitoylated.
Taken together these observations strongly
argue for the highly ordered lipid bilayer microenvironment as the sole
determinant of hydrophobic TC II-R monomers to dimerize in tissue
plasma membranes.
In conclusion, the results of the present study have shown that the dimerization of TC II-R, an important nutrient receptor, is a post-microsomal event and is due to its strong interactions with a highly ordered lipid bilayer membrane. The membrane fluidity regulated dimerization of plasma membrane TC II-R noted in this study may represent an unique situation. Other factors such as ligand binding (40) and phosphorylation-dephosphorylation (41) are known to regulate the oligomerization of several receptors. Finally, it is not known what physiological advantage a cell might have with TC II-R dimers in their plasma membranes. However, because the dimers are functional in ligand binding and bind 2 mol of ligand/dimer, it is likely that the existence of dimers will help in the rapid uptake of circulatory Cbl. Further studies are needed to identify the hydrophobic regions of TC II-R that mediate its interaction with the lipid bilayer, and such studies are possible once the sequence of TC II-R is known.