(Received for publication, April 4, 1995; and in revised form, June 14, 1995)
From the
Unfolded forms of the H2b subunit of the human
asialoglycoprotein receptor, a galactose-specific C-type lectin, are
degraded in the endoplasmic reticulum (ER), whereas folded forms of the
protein can mature to the cell surface (Wikström,
L., and Lodish, H. F.(1993) J. Biol. Chem. 268,
14412-14416). There are eight cysteines in the exoplasmic domain
of the protein, forming four disulfide bonds in the folded protein. We
have constructed double cysteine to alanine mutants for each of the
four disulfide bonds and examined the folding and metabolic fate of
each of the mutants in transfected 3T3 fibroblasts. We find that
mutation of the two cysteines nearest to the transmembrane region (C1)
does not prevent proper folding of the protein, whereas mutations of
the other three disulfides prevent proper folding of the protein and
all of the mutant proteins are degraded in the ER. A normal (20%)
fraction of the C1 mutant protein exits the endoplasmic reticulum and
is processed in the Golgi complex, and it does so at a faster rate
compared to the wild-type. Furthermore, the folded form of this mutant
protein is more resistant to unfolding by dithiothreitol than the
wild-type. The C1 mutant protein is expressed on the cell surface and
can form a functional receptor with the H1 subunit with similar binding
affinities for natural ligands as that of the wild-type receptor. The
same fraction of newly made mutant and wild-type proteins (
80%)
remain in the ER, but the mutant protein is degraded more quickly.
Thus, the presence of the C1 disulfide bond in the wild-type receptor
both reduces the rate of protein folding and exit to the Golgi and
slows the rate of ER degradation of the portion (
80%) of the
receptor that never folds properly.
The asialoglycoprotein (ASGP) ()receptor, also known
as the hepatic lectin, is a type II integral membrane protein that is
normally expressed only on mammalian hepatocytes. This
Ca
-dependent lectin binds to terminal galactose
residues on sugar side chains; these ligands are removed from the
circulation by receptor-mediated endocytosis via coated pits and
degraded in lysosomes(2, 3, 4) . The
functional human ASGP receptor is a hetero-oligomer consisting of two
types of subunits, H1 and H2, with a minimum stoichiometry of
(H1)
(H2)
(5) . H1 and H2 are 60%
homologous in amino acid sequence(6, 7) , and both
subunits are required for a functional
receptor(8, 9) . The polypeptide chain of each subunit
consists of four main domains: a short cytosolic amino-terminal
segment, a single hydrophobic transmembrane segment that also functions
as an uncleaved signal anchor sequence, followed by an exoplasmic
``stalk'' domain and, at the very carboxyl terminus, the
Ca
-dependent carbohydrate recognition domain. There
are two subtypes of H2: H2a and H2b, which differ only by the presence
of five extra amino acids in H2a near the transmembrane region on the
exoplasmic side that results from alternative splicing. In HepG2 human
hepatoma cells, most of the H2 expressed on the cell surface is
H2b(10) . In addition to the human receptor, highly homologous
ASGP receptors with identical functions have also been cloned and
analyzed in rat (11) and mouse(12) . The ASGP receptor
belongs to a family of animal proteins known as the C-type lectins with
the characteristic of requiring calcium ions for ligand
binding(13) . All members of these family share sequence
homology in the carbohydrate recognition domain. Within this domain of
about 130 amino acids, 18 invariant and 32 conserved residues are found
in all members(13) .
The important role of disulfide bonds
in determining and maintaining tertiary and quaternary structures of
secreted proteins and exoplasmic domains of membrane proteins has long
been recognized. These proteins form disulfide bonds between cysteine
residues due to the more oxidative redox state of the endoplasmic
reticulum (ER) compared to the cytosol(14) . The formation of
disulfide bonds in newly synthesized proteins occurs within the ER and
is catalyzed by the ER-resident enzyme protein disulfide
isomerase(15) . Intracellular disulfide-bonded folding
intermediates of the human chorionic gonadotropin subunit have
been identified(16) , and disulfide-bonded in vivo folding intermediates have also been reported for influenza
hemagglutinin(17) , retinol-binding protein(18) , and
the ASGP receptor H1 subunit(19) . Studies on the influenza
hemagglutinin have shown that all the disulfide bonds are required for
proper folding and protein maturation(20) . When cells are
treated with a reducing agent, dithiothreitol (DTT), which disrupts
disulfide bonds, newly made proteins that contain disulfide bonds, e.g. albumin and the ASGP receptor, are retained within the
ER, whereas proteins that do not have disulfides are secreted normally
in the presence of DTT(21) .
Depletion of Ca in the ER by treating cells with Ca
pump
inhibitors or Ca
ionophores also blocks exit of the
ASGP receptor from the ER and unfolded proteins
accumulate(1, 19) . Therefore, proteins that are
misfolded or cannot attain a properly folded state in the ER cannot
exit this organelle to the Golgi complex and are ultimately degraded in
the ER (22, 23) .
When expressed without H1 in transfected fibroblasts, about 80% of ASGP receptor H2b subunits are degraded in the ER(10, 24, 25) . The unfolded portion of the newly made H2b protein is selectively degraded within the ER, whereas the 20% that is folded matures to the cell surface(1) . Therefore, we felt it important to analyze further the relationship between folding and degradation of H2b within the ER. In this study, we determine the importance of each of the four disulfide bonds in the folding and degradation of the H2b protein by specifically mutating each pair of cysteine residues and observing the metabolic fate of the mutant proteins in transfected fibroblasts. We find that disruption of three of the disulfide bonds results in the inability of the protein to fold properly, and these cannot exit the ER and are degraded. However, mutation of the two cysteines nearest the transmembrane region (C1 mutation) actually causes the protein to fold faster than the wild-type and the mutant protein exits the ER to the Golgi complex more rapidly than does the wild-type. Furthermore, the C1 H2b mutant will form a functional receptor with H1 and bind a natural ligand with similar affinity to the wild-type receptor.
Figure 1: Schematic diagram of putative exoplasmic disulfide bonds of the human asialoglycoprotein receptor H2b subunit. C, cysteine residues in the exoplasmic domain; M, methionine residues; Cyto, cytosolic domain; TM, transmembrane domain; Exo, exoplasmic domain; numbers above the cysteine and methionine residues refer to their position in the amino acid sequence; C1-C4 refer to the putative disulfide bonds. Asterisk indicates position of N-linked glycosylation sites. Diagram is not to scale.
To determine if a disulfide is formed between these two cysteines
(referred to as the C1 disulfide), we take advantage of the methionine
residue at position 159, between these two cysteines, that is only one
of two methionines in the whole molecule, excluding the translation
initiation residue site. Met is also the only methionine
in the exoplasmic domain (Fig. 1). The mature complex
glycosylated H2b protein is
50 kDa, with a core protein mass of
35 kDa and three N-linked sugar side chains adding
5
kDa each to the total mass. In the experiment leading to Fig. 2,
proteins in H2b-transfected fibroblasts were reacted with iodoacetamide
to block free sulfhydryl groups and then radioiodinated with the
membrane-impermeable Bolton-Hunter reagent (sulfo-SHPP). The cells were
then dissolved in detergent and immunoprecipitated with an antiserum
against the carboxyl terminus of the H2 protein. We chose to analyze
only the mature, cell surface form of the H2b protein to avoid
ambiguities that can arise in the intracellular forms, which may not be
fully folded or are misfolded with non-native disulfides. If there is a
C1 disulfide bond, on digestion of the immunoprecipitated mature cell
surface H2b protein by cyanogen bromide, which specifically cleaves
after methionine residues, a
27-kDa fragment (with two N-linked sugar side chains) and a
17-kDa fragment (with
one N-linked sugar side chain), should be detected only in
reducing conditions but not under non-reducing conditions. Fig. 2shows the 50-kDa full-length complex glycosylated H2b
expressed on the cell surface of stably transfected 3T3 fibroblasts
analyzed under non-reducing (lane1) and reducing (lane3) conditions. After digestion with cyanogen
bromide, a portion of the 50-kDa protein is decreased in size to about
44 kDa due to cleavage at Met
but no smaller fragments
were observed under non-reducing conditions (lane2).
However, under reducing conditions, a
27-kDa fragment (which
corresponds to the carboxyl-terminal fragment with two N-linked sugar side chains) and a
17-kDa fragment (which
corresponds to the amino-terminal fragment with one N-linked
sugar side chain) were observed (lane4). These were
not observed under non-reducing conditions (lane2).
These observations strongly suggest that there is a reducible
intrachain disulfide linking Cys
and Cys
.
It is possible that the C1 disulfide bond is actually between
Cys
and Cys
, whereas the C2 disulfide is
between Cys
and Cys
, but the main
conclusion is that Cys
is disulfide-bonded. In summary,
the intrachain disulfides of the H2b protein are presumed to be:
Cys
/Cys
(C1), Cys
/Cys
(C2), Cys
/Cys
(C3), and
Cys
/Cys
(C4).
Figure 2: Cyanogen bromide digestion of H2b protein. Fibroblasts expressing wild-type H2b were treated with 0.1 M iodoacetamide in phosphate-buffered saline on ice for 5 min, after which cell surface proteins were radioiodinated with the water-soluble Bolton-Hunter reagent as described under ``Experimental Procedures.'' Cells were lysed and immunoprecipitated with an antiserum against the carboxyl terminus of the H2 protein, and the immunoprecipitated H2b protein was reacted in 70% formic acid with (lanes2 and 4) or without (lanes1 and 3) 50 mg/ml cyanogen bromide for 12 h. After the reaction, the proteins were subjected to SDS-PAGE under non-reducing (lanes1 and 2) or after reduction with 50 mM DTT (lanes3 and 4).
Figure 3:
Metabolic fate of H2b wild-type and
cysteine mutant proteins in stably transfected 3T3 fibroblasts. 3T3
cells expressing wild-type (lanes 1-6) or mutant H2b
proteins (C1 mutant, lanes 7-12; C2 mutant, lanes
13-18; C3 mutant, lanes 19-24; C4 mutant, lanes 25-30) were pulse-labeled with 0.3 mCi/ml
[S]Cys for 15 min (lanes1, 7, 13, 19, and 25) and then chased
in unlabeled medium for up to 4 h (lanes 2-6, 8-12, 14-18, 20-24, and 26-30). Cell lysates from various times of chase were
immunoprecipitated with an antiserum against the carboxyl terminus of
the H2 protein and then subjected to SDS-PAGE under reducing
conditions. Openarrow indicates the high mannose
precursor form of the protein. Solidarrow indicates
the complex glycosylated form of the
protein.
Figure 4: Kinetics of ER degradation and Golgi processing of H2b wild-type and cysteine mutant proteins in transfected 3T3 fibroblasts. The 43-kDa high mannose precursors and 50-kDa complex glycosylated forms of the H2b wild-type or mutant proteins in the fluorograms from Fig. 2were quantitated by scanning densitometry, normalized to amount of precursor after each pulse, and plotted against time of chase. Panel a, rate of loss of high mannose forms and appearance of complex glycosylated forms of wild-type protein. Panel b, rate of loss of high mannose forms and appearance of complex glycosylated forms of C1 mutant protein. Panel c, rate of loss of high mannose forms of C4 mutant protein.
Fig. 3(lanes 14-18, 20-24, and 26-30) shows that the C2, C3
and C4 mutants do not form any detectable complex glycosylated mature
forms. All the pulse-labeled proteins remain as 43-kDa forms (Endo
H-sensitive, data not shown). A 55-kDa polypeptide that appears in
all lanes is unlikely to be a processed form of the mutant H2b
receptor, since it is present in the pulse-labeled samples and its
mobility is not affected by N-glycanase digestion (data not
shown). We do not know the identity of this co-immunoprecipitated
protein. The immunoprecipitated 35-kDa polypeptide that appears in the
chase periods comprises the carboxyl terminus of the H2b protein; it is
produced by a proteolytic cleavage in the exoplasmic domain near the
membrane-spanning region (24, 25) . We consistently
observe that more of this fragment is produced in cells expressing the
C2, C3, and C4 mutants, consistent with the finding that no detectable
fraction of these proteins matures out of the ER. Fig. 4c shows that the kinetics of degradation of the Endo H-sensitive
form of the C4 mutant is similar to that of the wild-type. Similar
densitometric scans show that the C2 and C3 mutants also have similar
kinetics of degradation compared to the wild-type H2b (data not shown).
On non-reducing SDS-PAGE, less compact forms of a protein should experience more hydrodynamic resistance, and thereby migrate more slowly than more compact forms of the same protein. Formation of disulfide bonds in a protein should allow it to attain more compact forms and migrate faster under non-reducing conditions on SDS-PAGE than non-compact forms without disulfide bonds. Previous studies on folding of the ASGP receptor subunits showed folding intermediates that were separable by non-reducing SDS-PAGE(1, 19) . Fig. 5(lanes1 and 2) shows that, as judged by their differences in mobility on non-reducing SDS-PAGE, pulse-labeled H2b wild-type and C1 mutant proteins have a less compact and presumably unfolded structure (shadedarrow) but that they attain a more compact structure (lanes6 and 7, stripedarrow) after a 30-min chase period. However, if the cells are treated with 5 mM DTT for another 5 min at the end of the 30-min chase, a fraction of the wild-type H2b protein is ``unfolded'' to a less compact form (lane11), while almost all of the C1 mutant protein remains as the more compact species (lane12). Lanes 3-5 show that the C2, C3, and C4 mutants are also synthesized as less compact unfolded forms, similar to the wild-type (lane1, shadedarrow). After 30 min, the C2 and C4 mutants (lanes8 and 10) also attain a more compact or more folded form (stripedarrow) similar to the wild-type (lane6). However, most of the C3 mutant remains as the less compact unfolded form after 30 min (lane9). On treatment with 5 mM DTT, only a fraction of the wild-type is unfolded to the less compact form (lane11) but all of the C2, C3 and C4 proteins are unfolded to the less compact form (lanes 13-15). On reducing SDS-PAGE, all the bands migrate to the same positions (data not shown) and therefore results under non-reducing conditions reflect differential oxidative isoforms of the proteins. Experiments in which longer chase times were done (up to 3 h) showed identical results concerning the relative mobilities and DTT sensitivities of the wild-type and mutant proteins (data not shown). We conclude that the C2, C3, and C4 disulfides are required for proper folding of the protein. If any of these disulfides are missing, the protein cannot attain its properly folded form and cannot exit the ER and is all degraded within the ER. However, the absence of the C1 disulfide bond enhances protein folding and processing.
Figure 5:
Folding of H2b wild-type and cysteine
mutant proteins in transfected 3T3 fibroblasts. 3T3 cells expressing
wild-type (lanes1, 6, and 11) or
mutant H2b proteins (C1 mutant, lanes2, 7,
and 12; C2 mutant, lanes3, 8, and 13; C3 mutant, lanes4, 9, and 14; C4 mutant, lanes5, 10, and 15) were pulse-labeled with 0.3 mCi/ml
[S]Cys for 10 min (lanes 1-5) and
then chased in unlabeled medium for 30 min (lanes 6-15).
Where indicated, 5 mM DTT was added to the medium at the end
of the chase period for 5 min (lanes 11-15). All samples
were treated with 0.1 M iodoacetamide before the cells were
lysed. Cell lysates were immunoprecipitated with an antiserum against
the carboxyl terminus of the H2 protein, treated with N-glycanase to remove the sugar side chains, and then
subjected to SDS-PAGE under non-reducing conditions. Shadedarrow indicates the less compact form of the protein. Stripedarrow indicates the more compact form of the
protein.
Figure 6: Immunofluorescence localization of H2b wild-type and cysteine mutant proteins on the surface of transfected 3T3 fibroblasts. Live 3T3 cells expressing wild-type (a), C1 mutant (b), or C4 mutant (c) H2b protein were reacted at 4 °C first with a primary rabbit antibody against the carboxyl terminus of the H2 protein followed by a secondary Cy3-labeled goat anti-rabbit antibody. Cells were washed, fixed, and visualized by fluorescence microscopy.
A functional ASGP receptor requires both the H1 and H2 subunits, and it is possible that the C1 disulfide bond is required to form the functional receptor complex with H1. To determine if the H2b C1 mutant can form a functional receptor, we stably transfected cDNA encoding the H1 subunit into 3T3 fibroblasts expressing the wild-type or C1 mutant. Both cell lines showed specific and calcium-dependent binding to a well studied ligand for the ASGP receptor, human asialo-orosomucoid (data not shown), whereas cell lines expressing only the H2b wild-type or C1 mutant protein did not show any specific binding (data not shown). Fig. 7a shows the Scatchard plot of a binding study at 4 °C, employing cells expressing H1 and wild-type H2b, and radioiodinated asialo-orosomucoid. The results indicate a dissociation constant of 11 nM and about 600,000 surface receptors/cell. Fig. 7b shows the Scatchard plot of a similar binding study employing cells expressing H1 and C1 mutant H2b. The results indicate a binding constant of 9 nM and about 120,000 surface receptors/cell. These levels of expression are within the range of that exhibited by human hepatoma HepG2 cells (32) . The different levels of surface expression may be accounted for by different levels of H2 protein expression in the two cell lines used here. Pulse-labeling studies with the two cell lines show that they express equal levels of the H1 protein but that the wild-type H2b is expressed at about a 5-fold higher level than the C1 mutant (data not shown). However, both cell lines show specific ligand binding and similar binding affinities; the values are similar to those of wild-type receptors naturally expressed in cultured HepG2 cells(32) .
Figure 7:
Saturation binding of I-asialo-orosomucoid to transfected 3T3 fibroblasts
expressing H1 and wild-type H2b proteins (a) and H1 and C1
mutant H2b proteins (b). Cells were incubated with various
concentrations of radiolabeled ligand for 2 h at 4 °C with or
without excess unlabeled ligand. Total and nonspecific binding were
measured as described under ``Experimental Procedures,'' and
their difference, the specific binding, was analyzed by Scatchard
plots. B, specific bound ligand; B/F, ratio of
specific bound ligand to free ligand. K
,
dissociation constant.
Fig. 8(a and b) shows
that both the cell lines expressing H1 and wild-type H2b and the cell
lines expressing H1 and C1 mutant H2b are capable of endocytosis and
degradation of asialo-orosomucoid. With both cell lines,
cell-associated radioactivity plateaus after about 30 min of incubation
at 37 °C and degradation products appear in the medium within 30
min. The rates of total ligand uptake and degradation are similar in
both cell lines: 3.5 pg of ligand/min/µg of cell protein over
4 h at 37 °C. It is interesting that although the cell line
expressing H1 and H2b C1 receptors has about 5-fold fewer surface
receptors than one expressing H1 and wild-type H2b (as determined by
the saturation binding experiments), they show a similar rate of ligand
endocytosis and degradation. This may indicate that the turnover rate
of the endocytic cycle of the mutant receptor is higher than that of
the wild-type receptor.
Figure 8:
Uptake and degradation of I-asialo-orosomucoid by transfected 3T3 fibroblasts
expressing H1 and wild-type H2b proteins (a) and H1 and C1
mutant H2b proteins (b). Cells were incubated with 2 µg/ml
radiolabeled ligand for various times at 37 °C. At each time point,
the medium was analyzed for
I degradation products and
the cell-associated
I was also determined as described
under ``Experimental
Procedures.''
We conclude that the absence of the C1 disulfide bond in the H2b protein does not prevent its transport to the cell surface; it can form a functional receptor when co-expressed with the H1 subunit. This receptor binds to a natural ligand, asialo-orosomucoid, with an affinity similar to that of the wild-type receptor. The mutant receptor can also perform its natural functions of receptor-mediated endocytosis, ligand degradation, and presumably receptor recycling. Therefore, it is unlikely that the these two cysteines are required for the formation of a functional receptor complex.
A large fraction of the ASGP receptor H2b subunit expressed in transfected fibroblasts in the absence of the H1 subunit is degraded in the ER. Depletion of calcium ions in the ER causes all of H2b to be retained in a misfolded state in the ER and eventually is degraded there(1) . When cells expressing the H1 subunit are depleted of calcium ions or treated with DTT, all of the H1 protein is also retained in and degraded in the ER(19) . Studies on the T-cell receptor subunits also showed that the ER degradation process is enhanced in the presence of DTT (38) or depletion of calcium ions(39) , conditions that prevent proper folding of proteins. Therefore, proper folding and formation of disulfide bonds in secretory or membrane proteins are normally required for transport out of the ER.
As we expected, three of the disulfide bonds (C2, C3, and C4) in the
H2b protein are essential for proper folding of the protein as absence
of any one of them prevents transport of the protein out of the ER to
the Golgi and all of the newly made protein is degraded in a pre-medial
Golgi compartment. As judged by non-reducing SDS-PAGE and sensitivity
to DTT, the C2, C3, and C4 mutants cannot fold as well as the wild-type
protein, as only the wild-type protein can attain resistance to DTT
unfolding (although only a fraction of it can do so). These
observations confirm the previous deduction that only folded forms of
the H2b protein can exit the ER to the cell surface(1) .
Presumably, a folded form of the H2b protein that is resistant to DTT
unfolding, a conformation attained by a fraction (20%) of the
wild-type protein, is the prerequisite for ER to Golgi transport and
appearance on the plasma membrane. An interesting result from these
assays is that the C3 mutant cannot attain any compact structure after
a 30-min chase period, whereas the C2 and C4 mutants can attain some
compact structure(s), although these are all sensitive to DTT unfolding (Fig. 5). This suggests that the C3 disulfide bond plays a more
important role in attaining or maintaining a folded structure of the
H2b protein than do the C2 and C4 disulfide bonds, although the C3
disulfide bond is formed by two cysteines that are very close in amino
acid sequence positions (Cys
and Cys
)
relative to the C2 and C4 disulfide bonds.
Surprisingly, mutation of
the two cysteines nearest to the transmembrane region to alanine
residues does not prevent maturation of the protein to the cell
surface. In contrast, the mutant protein is folded more rapidly than
the wild-type, as all of the mutant protein that attains a compact
structure (as judged by mobility on non-reducing SDS-PAGE) within 30
min is resistant to unfolding by DTT, whereas less than half of the
compact form of the wild-type protein is resistant to DTT unfolding.
Furthermore, the mutant protein is processed more rapidly than the
wild-type in that it is transported to the Golgi complex more quickly.
However, the proportion of the mutant protein that becomes
Golgi-processed (20%) is about the same as that of the wild-type.
The portion of the mutant H2b protein that remains in the ER is
degraded at a higher rate than the wild-type.
We speculate that, when present, the C1 cysteines cause the protein to be misfolded and to be bound by chaperones in the ER such as protein disulfide isomerase or BiP (heavy chain binding protein). The binding would cause the protein to remain in the ER for longer periods, during which a fraction of them may refold to form the native disulfide bonds after which the protein is exported to the Golgi complex. As a result, ER to Golgi transport of the protein is delayed and degradation in the ER is also slowed down. In the C1 mutant, these cysteines are absent. The misfolded proteins would not be formed, and the protein would not bind chaperones. Therefore, the C1 mutant would fold faster and exit the ER quicker, while the fraction that is unable to fold would remain in the ER and would be quickly degraded. This hypothesis indicates that binding of H2 to certain chaperones might delay both folding and ER degradation. It is consistent with our observation that the C2, C3, and C4 mutant proteins are degraded at the same rate as the fraction of the wild-type that remains in the ER, as all these proteins would be misfolded and bound to chaperones. We have no direct evidence for this hypothesis, as we have not been able to detect binding of specific chaperones to the wild-type or mutant H2b proteins.
We have demonstrated previously
that degradation of the H2 subunit in the ER occurs via two pathways,
one which involves an initial cleavage of the protein (most likely by
signal peptidase) to form a 35-kDa carboxyl-terminal fragment as a
degradation intermediate, and another pathway which does not go through
this intermediate. The pathway that is not dependent on the formation
of the 35-kDa intermediate is inhibited by the protease inhibitors TLCK
or TPCK and is probably the major pathway(25) . In the presence
of TLCK or TPCK, unfolded forms of the H2b protein accumulate in the
ER, therefore suggesting that it is the unfolded forms of H2b that are
degraded in the ER(1) . The degradation of the C1 mutant
protein in the ER is also inhibited in the presence of TLCK or
TPCK(); this is consistent with the notion that the unfolded
fraction of the C1 mutant protein that remains in the ER is degraded
via the major TLCK and TPCK-sensitive degradation pathway. Relative to
wild-type H2b, more of the 35-kDa carboxyl-terminal fragment was
produced from the C2, C3 and C4 mutants. Its higher abundance may
indicate that a larger fraction of each of these mutants is degraded
via the pathway that goes through this intermediate rather than the
pathway that does not involve cleavage to the 35-kDa fragment.
We
find that the H2b C1 mutant protein is capable of forming a functional
receptor when co-expressed in fibroblasts with the wild-type H1
subunit. The H1/H2b C1 mutant receptor binds a natural ligand with an
affinity similar to that of the wild-type receptor and is capable of
catalyzing ligand endocytosis and degradation. The rate of endocytosis
and recycling of the mutant receptor actually appears to be slightly
higher than the wild-type. This may indicate that the mutant protein
exhibits a higher rate of turnover in the secretory/endocytic pathway.
Another possibility is that access to the coated-pits is the
rate-limiting step for rate of endocytosis and that this process is
saturated at low numbers of receptors. Therefore, the rate of
endocytosis and recycling would be similar for the wild-type and the C1
mutant even though they have a 5-fold difference in the number of
cell-surface receptors. Importantly, the two C1 cysteines in the H2
protein are not required for proper receptor function. In contrast,
studies on the influenza hemagglutinin have shown that all the
disulfide bonds are required for proper folding and secretion of the
protein(20) . However, when certain cysteine residues are
mutated in the subunit of human chorionic gonadotropin (40) and human lysozyme (41) , the rate of secretion is
enhanced. Our study also demonstrates that loss of a disulfide linkage
can lead to enhanced folding and formation of a fully functional
protein.
The presence of the C1 cysteines in the wild-type ASGP receptor H2 subunit may hinder protein folding and processing. That they have been selected in evolution suggests that higher efficiency in folding and processing may not always be advantageous. Perhaps the presence of the C1 cysteines serves to reduce the turnover rate of a receptor whose expression need not be highly regulated. Nevertheless, it would be interesting to study the role of the C1 cysteines during the folding of the protein by determining and comparing the folding pathways of the wild-type and C1 mutant proteins.