(Received for publication, July 28, 1995)
From the
The transmembrane 4 (TM4) superfamily contains many important leukocyte differentiation-related surface proteins including CD9, CD37, CD53, and CD81; tumor-associated antigens including CD63/ME491, CO-029, and SAS; and a newly identified metastasis suppressor gene R2. Relatively little is known, however, about the structure and aggregation state of these four transmembrane-domained proteins. The asymmetrical unit membrane (AUM), believed to play a major role in stabilizing the apical surface of mammalian urothelium thus preventing it from rupturing during bladder distention, contains two TM4 members, the uroplakins (UPs) Ia and Ib. In association with two other (single transmembrane-domained) membrane proteins, UPII and UPIII, UPIa and UPIb form 16-nm particles that naturally form two-dimensional crystalline arrays, thus providing unique opportunities for studying membrane structure and function. To better understand how these proteins interact to form the 16-nm particles, we analyzed their nearest neighbor relationship by chemical cross-linking. We show here that UPIa and UPIb, which share 39% of their amino acid sequence, are cross-linked to UPII and UPIII, respectively. We also show that UPIa has a propensity to oligomerize, forming complexes that are stable in SDS, and that UPII can be readily cross-linked to form homodimers. The formation of UPII homodimers is sensitive, however, to octyl glucoside that can solubilize the AUMs. These data suggest that there exist two types of 16-nm AUM particles that contain UPIa/UPII or UPIb/UPIII, and support a model in which the UPIa and UPII occupy the inner and outer domains, respectively, of the UPIa/UPII particle. This model can account for the apparent ``redundancy'' of the uroplakins, as the structurally related UPIa and UPIb, by interacting with different partners, may play different roles in AUM formation. The model also suggests that AUM plaques with different uroplakin compositions may differ in their assembly, and in their abilities to interact with an underlying cytoskeleton. Our data indicate that two closely related TM4 proteins, UPIa and UPIb, can be present in the same cell, interacting with distinct partners. AUM thus provides an excellent model system for studying the targeting, processing, and assembly of TM4 proteins.
The ``transmembrane four'' (TM4) ()superfamily is a recently described gene family that
encodes a group of cell surface proteins all possessing four conserved
transmembrane domains. Members of this family are found in lymphocytic,
mesenchymal, and epithelial cells (reviewed by Horejsi and Vlcek(1991)
and Wright and Tomlinson(1994)). Thus the TM4 proteins that have been
identified so far include several tumor-related surface proteins CO-029
(Szala et al., 1990), L6 (Marken et al., 1992), SAS
(Jankowski et al., 1994), and R2 (Gaugitsch et al.,
1991) (the last was recently described as a metastasis suppressor gene
for prostate cancer (Dong et al., 1995));
leukocyte-differentiation markers CD9 (Boucheix et al., 1991;
Lanza et al., 1991), CD37 (Classon et al., 1990),
CD53 (Amiot, 1990; Korinek and Horejsi, 1993), CD63 (Hotta et
al., 1988; Metzelaar et al., 1991), CD81 (also known as
TAPA-1; Oren et al., 1990; Engel and Tedder, 1994), and CD82
(Lebel et al., 1994; Nagira et al., 1994); as well as
major epithelial differentiation products uroplakins Ia and Ib (Yu et al., 1994). Closely related molecules, SM23 and SJ23, have
even been found in parasitic helminth schistosomes (Davern et
al., 1991; Reynolds et al., 1992), indicating that
members of this gene family are conserved during evolution. Since the
intron positions of several of the TM4 genes are conserved, these genes
may have diverged from a common ancestral gene (Horejsi and Vlcek,
1991; Wright et al., 1993; Wright and Tomlinson, 1994). Recent
data indicate that some of these TM4 proteins may play important roles
in cell growth, adhesion, and metastasis (Horejsi and Vlcek, 1991;
Miyake et al., 1991; Schick and Levy, 1993; Wright and
Tomlinson, 1994; Dong et al., 1995). However, many crucial
questions regarding the structure and function of TM4 proteins remain
unanswered. For example, how do these integral membrane proteins, most
of them lacking a significant cytoplasmic domain, perform their
functions on the cell surface? In several cases, more than one TM4
protein exists in the same cell; thus CD81 and CD82 coexist in T cells
(Imai and Yoshie, 1993; Nagira et al., 1994), and uroplakins
Ia and Ib coexist in the differentiated urothelial cells (Yu et
al., 1994). In such cases, do these TM4 proteins always interact
with each other? Or do some of these TM4 proteins interact specifically
with other integral membrane proteins?
Uroplakins Ia (UPIa; 27-kDa) ()and Ib (UPIb; 28-kDa; also known as TI-1) (Kallin et
al., 1991) are two newly identified TM4 proteins that are
synthesized by the terminally differentiated, superficially located
cells of mammalian urothelium (Yu et al., 1994). Together with
two other proteins, i.e. the 15-kDa uroplakin II (Lin et
al., 1994, 1995) and the 47-kDa uroplakin III (Wu and Sun, 1993),
the UPIa and UPIb are major protein components of the so-called
asymmetrical unit membrane (AUM) (Wu et al., 1990; Yu et
al., 1990), which forms numerous plaques covering about 80% of the
apical surface area of mammalian urothelium (Porter and Bonneville,
1963; Hicks, 1965; Porter et al., 1967; Koss, 1969; Chlapowski et al., 1972). These AUM plaques are believed to play a role
in stabilizing the luminal surface of the epithelium thus preventing it
from rupturing during bladder distention (Staehelin et al.,
1972; Minsky and Chlapowski, 1978; Sarikas and Chlapowski, 1986).
Recent biochemical data indicate that the major hydrophilic loop
interconnecting the third and fourth trans-membrane domains of UPIa and
UPIb is exposed on the extracellular surface, because this domain
becomes protected from protease digestion once the in vitro transcribed and translated UPIa is inserted into dog pancreatic
microsomes (Yu et al., 1994). In addition, in both UPIa and
UPIb, this domain contains an N-glycosylation site which
harbors high mannose type carbohydrates (Wu et al., 1994; Yu et al., 1994). These data strongly suggest that UPIa and UPIb,
like several other members of the TM4 superfamily, assume the so-called
``type III'' transmembrane configuration with the major
hydrophilic domain extending into the extracellular space leaving very
little cytoplasmic domains (Yu et al., 1994).
The asymmetrical unit membrane offers unique opportunities for studying the detailed structural arrangement and possible function of the two differentiation-dependent members of the TM4 family, i.e. the uroplakins Ia and Ib, because AUMs can be purified in milligram quantities (Wu et al., 1990; 1994). Moreover, uroplakins interact closely with one another forming highly organized 16-nm protein particles that naturally form two-dimensional crystalline arrays thus greatly facilitating a detailed analysis of protein structure (Hicks and Ketterer, 1969; Vergara et al., 1969; Chlapowski et al., 1972; Taylor and Robertson, 1984; Walz et al., 1995). We have therefore probed the topographical relationship among the four major integral membrane protein subunits of the asymmetrical unit membrane using the chemical cross-linking approach. Unexpectedly, our results indicate that uroplakins Ia and Ib are cross-linked to the 15-kDa uroplakin II and the 47-kDa UPIII, respectively. The fact that the two structurally related uroplakins Ia and Ib are cross-linked to different partners suggests that the two TM4 proteins play distinct roles in AUM structure. In addition, we present data showing that, in intact AUMs, uroplakin II can be cross-linked to form a homodimer, and that UPIa can form oligomers that are stable in SDS. Taken together, these results suggest a model in which uroplakins Ia and II occupy the inner and outer domains, respectively, of a 16-nm protein particle, and raise the possibility that AUMs are composed of two types of 16-nm particles containing different subsets of uroplakin molecules.
A prerequisite of this approach was the availability of antibodies that were monospecific for individual uroplakin molecules. We therefore raised a panel of rabbit antisera against synthetic peptides corresponding to sequences of the four major uroplakins. Immunoblotting established that all these antisera reacted strongly with their respective uroplakins (Fig. 1). Antisera to uroplakins Ia, II, and III recognized well defined 27-, 15-, and 47-kDa protein bands, respectively (Fig. 1b). Antisera to uroplakin Ib recognized multiple bands in the molecular mass range of 25 to 28 kDa; however, this apparent heterogeneity could be completely accounted for by glycosylation (Wu et al., 1994; Yu et al., 1994). Moreover, at least one antiserum for each uroplakin was shown to recognize, specifically, only the corresponding uroplakin in the crude urothelial membrane fraction (Fig. 1).
Figure 1:
Specificity of antibodies against
individual bovine uroplakins. a, proteins of crude bovine
urothelial membranes (lane 1), Sarkosyl-washed AUMs (lane
2), and additionally NaOH-washed AUMs (lane 3) were
dissolved in 1% SDS at room temperature, resolved by SDS-PAGE, and
visualized by silver nitrate (AgNO)
staining. b, proteins of crude urothelial membranes (odd-numbered lanes) and Sarkosyl-washed AUMs (even-numbered lanes) were electrophoretically transferred to
nitrocellulose and immunoblotted using (lanes 1 and 2) antibodies against a synthetic oligopeptide of UPIa; (3 and 4) anti-UPIb; (5 and 6) anti-UPII; (7 and 8) another anti-UPII; and (9 and 10) anti-UPIII. For the sequences of the synthetic
oligopeptides, see ``Materials and Methods.'' Numbers on the left denote the molecular weights (M.W.) of standard
proteins. The relative positions of the four major uroplakins (the
27-kDa UPIa, the 28-kDa UPIb, the 15-kDa UPII, and the 47-kDa UPIII)
are marked on the right. Note that most of the antibodies are
monospecific for their respective uroplakin antigens (see
text).
These monospecific antisera enabled us to monitor the cross-linking status of the uroplakins that were present in crude urothelial membranes that had been treated with various concentrations of EGS (Fig. 2). This experiment revealed the formation of three well defined, cross-linked uroplakin species. A new 22-kDa band was recognized only by the uroplakin II antibody and was therefore presumably a UPII homodimer; a 35-kDa band was recognized by antisera to both UPII and UPIa and was thus likely a heterodimer of UPIa and UPII; and finally a 72-kDa band reacted with antisera to both UPIb and UPIII, suggesting a UPIb/UPIII heterodimer (Fig. 2). Similar results were obtained using a hydrophilic analogue of EGS, the sulfo-EGS, although the yield of the UPIa/UPII heterodimer was greatly reduced (Fig. 3).
Figure 2: Chemical cross-linking of uroplakins that are present in native urothelial membranes. Crude bovine urothelial membranes were cross-linked with (lane 1) 0, (2) 0.5, (3) 1, (4) 2, (5) 3, (6) 4, and (7) 5 mM EGS. Their proteins were dissolved in 1% SDS, resolved by SDS-PAGE, electrophoretically transferred to nitrocellulose, and then immunoblotted with (a) antibodies against UPIa (anti-UPIa), (b) anti-UPII, (c) anti-UPIb, and (d) anti-UPIII. Note in Panels a) and b the formation of a cross-linked 35-kDa band that was recognized by both anti-UPIa and anti-UPII (labeled Ia/II), and a 22-kDa band recognized only by anti-UPII (labeled II/II). Also note in Panels c and d the formation of a 72-kDa band recognized by both anti-UPIb and anti-UPIII (labeled Ib/III), a 66-68-kDa band recognized by anti-UPIb, and a 74-kDa band recognized by anti-UPIII. The relative positions of molecular weight (M.W.) standards, as well as those of various uroplakin monomers and dimers are shown on the left and right, respectively.
Figure 3: Cross-linking of crude urothelial membranes using hydrophobic versus hydrophilic bifunctional cross-linking reagents. Crude bovine urothelial membranes were cross-linked with (a) EGS and (b) its hydrophilic analog, sulfo-EGS (S-EGS). The proteins of these cross-linked membranes were separated by SDS-PAGE and subjected to immunoblotting using anti-UPIa and anti-UPII, as indicated. Note that these two reagents are equally effective in generating the 22-kDa UPII homodimer (II/II); however, only the hydrophobic EGS yielded the UPIa/II heterodimer. The positions of various uroplakin monomers and dimers are marked on the sides.
Figure 4: Chemical cross-linking of uroplakins that are present in purified AUMs. Sarkosyl-washed AUMs (lanes 1 and 3) and the AUMs that had been further washed with NaOH (lanes 2 and 4) were incubated with only a buffer (lanes 1 and 2) or with 5 mM EGS (lanes 3 and 4). Proteins of these membranes were immunoblotted with (a) anti-UPIa and with (b) anti-UPII. Note the generation of the UPII/UPII homodimer, the UPIa/UPII heterodimer, and a UPIa/UPIa homodimer, thus confirming the crude membrane results. A 65-kDa species (?) may represent a UPIa oligomer (see Fig. 5a and text).
Figure 5:
Identification of the cross-linked
uroplakin complexes by two-dimensional, diagonal gel electrophoreses.
The proteins of EGS cross-linked AUMs were dissolved in 1% SDS and
separated by first dimensional SDS-PAGE. After staining with Coomassie
Blue and destaining, individual gel lanes were excised, incubated in 1 M hydroxylamine to cleave the cross-linked species, and
subjected to a second dimensional (slab) SDS-PAGE. The two-dimensional
gels were then (a) stained with silver nitrate (AgNO), or immunoblotted with (b) anti-UPIa or (c) anti-UPII. Lanes 1 and 2 are side lanes showing the proteins of either control AUMs (lane 1) or EGS cross-linked AUMs (lane 2) that were
resolved only during the second dimensional SDS-PAGE. Arrows marked with 1 and 2 denote the directions of the
first and second dimensional SDS-PAGE. The molecular weights (MW) of the marker proteins are indicated on the right of Panel a. Note the cleavage of a 35-kDa cross-linked
protein (lane 2), giving rise to a 27-kDa UPIa and a 15-kDa
UPII (dashed lines). Also note the cleavage of a 22-kDa
cross-linked protein yielding a 15-kDa UPII (dotted lines). A
series of UPIa-related spots (horizontal arrows), that can be
seen above the diagonal in Panels a and b, represent
oligomerized UPIa (see text).
So far we
identified the cross-linked uroplakin species based on their relative
sizes and their immunoreactivities with various antibodies to
uroplakins. To confirm these assignments, we resolved the cross-linked
AUM proteins by SDS-PAGE, cut out the entire gel lane containing the
cross-linked uroplakins, cleaved them by incubating the gel strip in 1 M NHOH, and resolved the released uroplakins by a
second dimensional SDS-PAGE. In this procedure, only the monomers that
were released from a cross-linked product during the hydroxylamine step
would migrate below the diagonal (Fig. 5a). Such an
analysis revealed the existence of a 35-kDa, EGS cross-linked species
which was cleaved by a hydroxylamine releasing a 27-kDa uroplakin Ia
plus a 15-kDa uroplakin II (Fig. 5; see the circled protein
spots connected by a dashed line), thus confirming the
identity of the UPIa/UPII heterodimer. The results also clearly
established the presence of a 22-kDa cross-linked product that, upon
hydroxylamine treatment, released only a 15-kDa uroplakin II, thus
confirming the identity of the uroplakin II homodimer (Fig. 5; dotted line). Finally, we observed a series of UPIa oligomers
of 48 kDa (dimer) and 70 kDa (trimer), which apparently were formed
during the hydroxylamine treatment (Fig. 5, a, horizontal arrows, and b).
Figure 6: Effects of detergents and chain length of the cross-linking reagents on uroplakin cross-linking. Cross-linking was carried out on total or crude membranes (a) or highly purified AUMs (b and c), using either EGS (16 Å; Panels a and b) or DFDNB (3 Å; Panel c). The cross-linked membrane proteins were dissolved in 1% SDS, resolved by SDS-PAGE, and immunoblotted using anti-UPIa or anti-UPII, as indicated. In Panels a and b, lanes 1 are controls without cross-linking. EGS cross-linking was carried out in 10 mM Hepes buffer (lanes 2), or in the same buffer containing 2% octyl glucoside (lanes 3) or 2% Triton X-100 (lanes 4). Note that the yield of UPIa/UPII heterodimer is not affected by the detergents (lanes 2-4); however, the formation of UPII homodimer was largely abolished by octyl glucoside (lanes 3), although unaffected by Triton X-100 (lanes 4; see Fig. 7for the scanning of these lanes). Also note, in Panel c, that the short armed DFDNB failed to produce the UPII homodimer, although it yielded the UPIa/UPII heterodimer. The relative positions of uroplakin monomer and dimer are indicated on the sides.
Figure 7: Octyl glucoside abolishes selectively the formation of uroplakin II homodimer. Lanes 2-4 of the immunoblots produced with anti-UPII, as shown in Fig. 6, a and b, were scanned for densitometry using a Universal Imaging Program. The samples correspond to (a) crude membranes and (b) purified AUMs that have been EGS cross-linked (1) without detergent(-), (2) with 2% octyl glucoside (O.G.), or (3) with 2% Triton X-100 (T.X.). The small, white arrow indicates the direction of SDS-gel electrophoresis, and the large, open arrows mark the positions of the UPII homodimer. Note that Triton had relatively little effect on uroplakin cross-linking, while octyl glucoside greatly reduced the formation of the UPII homodimer in both crude membrane and purified AUMs.
To assess the relative distance
of the cross-linked -lysine groups, we treated purified AUMs with
DFDNB, which has an arm length of only 3 Å (versus the
16 Å of EGS and sulfo-EGS). Like EGS, DFDNB cross-linked the
UPIa/UPII heterodimer and UPIa/UPIa homodimer. However, it failed to
cross-link the UPII/UPII dimer (Fig. 6c), suggesting
that the
-lysines in UPII/UPII cross-linking were >3 Å
apart.
We have probed the topographical relationship of the
uroplakins in the asymmetrical unit membrane using bifunctional
cross-linking reagents. The results that we have obtained so far have
several important features. First, we identified the same set of
cross-linked uroplakin dimers, regardless whether we used the purified
AUMs ( Fig. 4and Fig. 5), or the crude urothelial
membranes ( Fig. 2and Fig. 3), as our starting material.
This suggests that the topographical relationships that exist in the
relatively unperturbed, crude urothelial membranes must have been
maintained to a large extent in our purified AUMs. Second, the
cross-linking patterns were highly reproducible over a wide range of
experimental conditions covering different types and concentrations of
the cross-linking reagents (Fig. 2Fig. 3Fig. 4Fig. 5). Moreover, the
cross-linking was highly efficient capable of capturing >30% of the
uroplakin monomers using the reagent concentrations that we have tested (Fig. 2Fig. 3Fig. 4), thus making it less likely
that we are observing the cross-linking of uroplakins entrapped in a
minor AUM conformation. Third, the cross-linking of purified AUMs
resulted in the formation of only a few, major protein complexes that
have been identified as containing purely uroplakins. Thus the
cross-linking of UPIa with EGS yielded almost exclusively the
UPIaUPII complex, and UPII yielded predominantly the UPIa/UPII
heterodimer and the UPII homodimer (Fig. 2Fig. 3Fig. 4). Such a relatively simple
cross-linking pattern of the purified AUMs is perhaps to be expected,
given the fact that AUMs are known to contain only four major protein
subunits (Wu et al., 1990, 1994; Yu et al., 1994). It
was unexpected, however, that crude urothelial membranes yielded no
additional cross-linked species, although of course this negative
finding does not rule out additional protein:protein interactions that
may exist in situ. Taken together, our results strongly
suggest that the uroplakin pairs that we have identified so far by the
cross-linking approach reflect important protein:protein interactions
that occur in the asymmetrical unit membrane.
These results indicate that UPII is involved in two different kinds of protein:protein interactions. Its binding to UPIa is short-ranged as they can be cross-linked not only by EGS but also by the 3 Å DFDNB, and this binding is relatively strong as it is stable in octyl glucoside. In contrast, the binding of UPII to another UPII is relatively distant as the cross-linking required a long-armed reagent, and the binding is relatively weak as it can be disrupted by octyl glucoside. This raises the possibility that UPII interacts with UPIa within a 16-nm particle, but with UPII of perhaps another particle (see below). This also raises the possibility that a detergent's ability to break the UPII:UPII interaction, which may be involved in bridging the neighboring 16-nm particles (see below), enables the detergent to solubilize the AUMs.
With these questions in mind, it is interesting to note that the four uroplakins form two pairs as defined by the two known (cross-linked) heterodimers, i.e. UPIa/UPII and UPIb/UPIII. Each of these dimers consists of a four transmembrane-domained member (UPIa or UPIb) plus a single transmembrane-domained protein (UPII or UPIII). Although all of the uroplakins appear to be able to form oligomers (Fig. 2Fig. 3Fig. 4Fig. 5) (Wu and Sun, 1993; Wu et al., 1994), so far we have not found conditions under which we can cross-link UPIa to UPIII (instead of UPII), or UPIb to UPII (instead of UPIII), suggesting a specificity in uroplakin interaction that was not suspected previously. This specificity raises the possibility that AUMs, despite the fact that they appear to be morphologically homogeneous, may actually contain two distinct populations of 16-nm protein particles, one composed of UPIa and UPII, and another of UPIb and UPIII (Fig. 8).
Figure 8: A schematic model showing the possible existence of two types of AUM plaques containing 16-nm particles that are composed of (a) uroplakins Ia and II and (b) uroplakins Ib and III. a, in the UPIa/UPII model, the 27-kDa UPIa and the 15-kDa UPII are hypothesized to occupy the inner and outer domains of a 16-nm protein particle. This model can account for (i) the oligomerization of UPIa, (ii) the cross-linking of UPIa/UPII heterodimer, (iii) the cross-linking of UPII/UPII homodimer, and (iv) the selective disruption of the UPII homodimer formation by octyl glucoside. b, UPIb and UPIII occupy the inner and outer domains, respectively, of the UPIb/UPIII model. This model can account for the cross-linking of UPIb/UPIII heterodimer, as well as the efficient cross-linking of UPIb/UPIb and UPIII/UPIII homodimers. The stain-excluding map of bovine AUM was taken from Wu et al. (1994) (also see Walz et al.(1995)). For details, see the text.
This kind of consideration also suggests that the UPIa/UPII particles are not intermixed, within a single plaque, with the other kind of UPIb/UPIII particles, because if that were the case we should see the cross-linking of UPII of one particle to the UPIII of a neighboring particle, and we have not yet seen that. This raises the possibility that there are two types of urothelial plaques, one consists purely of 16-nm particles containing UPIa and UPII, while the other consists of particles containing UPIb and UPIII. This hypothesis is schematically depicted in a working model, shown in Fig. 8, that can account for all of the available data. This model is attractive because it can solve two puzzles. It can explain the redundancy of uroplakins, as the two TM4 family members, i.e. the UPIa and Ib, may actually interact with different partners and thus play related but distinct roles in AUM formation. This hypothesis can also solve the stoichiometry puzzle, because it now predicts a molar relationship of UPIa = UPII and UPIb = UPIII, thus allowing variations in the overall stoichiometry, depending on the ratio of the two types of AUM plaques. In addition, this model predicts that the two types of AUMs may play different biological roles in terminally differentiated urothethelial cells. For example, since of all the known uroplakins only the UPIII has a long cytoplasmic domain, this uroplakin may play a role in anchoring the AUM plaques into a cytoskeletal network (Wu and Sun, 1993). Is it then possible that only the UPIb/UPIII plaques can bind to the cytoskeleton? Since uroplakin II is the only AUM protein that has a long preprosequence, we need to consider the possibility that the UPII prosequence may be involved in regulating AUM assembly in the Golgi (Lin et al., 1994). Is it then possible that the assembly of the UPIa/UPII plaques is regulated differently from that of the other kind of plaques? Additional experiments are obviously needed to further study the possible heterogeneity of AUMs and to address some of the questions raised herein.
Imai and Yoshie(1993) have shown that CD81 and CD82, which coexist in T cells, can be coimmunoprecipitated, suggesting that they interact with each other forming a complex. Our finding that UPIa and UPIb, two members of the TM4 family, interact with different partners in AUM was therefore unexpected. Taken together, these data indicate that members of the TM4 family, although structurally related, have diverse structural and functional properties.