Selective Interactions of UPIa and UPIb, Two Members of the Transmembrane 4 Superfamily, with Distinct Single Transmembrane-domained Proteins in Differentiated Urothelial Cells

Abstract

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 crosslinked 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.

Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-Å resolution

“…5 A). Supporting this concept are the findings that UP Ia and Ib can be readily cross-linked with UP II and IIIa, respectively, to form heterodimers UP Ia/II and Ib/IIIa (Wu et al, 1995) and that the formation of the UP Ia/II and Ib/IIIa heterodimers is a prerequisite for their ER exit (Deng et al, 2002; Tu et al, 2002; Hu et al, 2005). There are two types of secondary interactions between the primary complexes in the UP tetraspanin network (Fig.…”

“…UP tetraspanin web consists of hexagonal arrays of 16-nm UP protein particles. Each of these particles contains two highly homologous (34% identical) tetraspanins, UP Ia and Ib, which pair specifically and stoichiometrically with their single-spanning transmembrane protein partners, UP II and UP IIIa, respectively (Wu et al, 1995; Liang et al, 2001; Deng et al, 2002; Tu et al, 2002; Min et al, 2003; Hu et al, 2005). The naturally occurring crystalline UP web forms rigid-looking plaques (urothelial plaques; also known as asymmetric unit membrane, or AUM) covering almost the entire apical surface of the mammalian urothelium (Staehelin et al, 1972; Hicks, 1975; Kachar et al, 1999).…”

Glycosylation of uroplakins. Implications for bladder physiopathology

“…In contrast, UPII and III (15-kDa and III 47-kDa, respectively) cross the membrane only once and their single transmembrane domain (Fig. 3) share a stretch of ~12 amino acid residues on the extracellular side [5, 8, 9, 20–22].

Fig.

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“…1b) are constructed by uroplakins (UPs) (Fig. 1c), the markers of the advanced degree of the differentiation stage of umbrella cells [reviewed in [5, 8–11]. In mammals characterized by a thicker layer of the urothelium ( e.g ., humans), UPs are mostly present on the cell surface, in mammals with thinner urothelium ( e.g .…”

Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization

“…Existing data indicate that UPIa and UPIb interact with proUP∥ (the immature form of UP∥ still containing its prosequence) and UPIIIa respectively to form UPIa/UP∥ and UPIb/UPIIIa heterodimers that can then exit from the ER (endoplasmic reticulum) [18,27–29]. We have shown previously that a monoclonal antibody, AE31, recognizes a composite proUPII epitope consisting of a segment of the prosequence (−50 to −40) and another of the mature sequence (+40 to +60), suggesting that proUPII adopts a hairpin-like structure [28].…”