SELECTIVE INTERACTIONS OF UPIA AND UPIB, 2 MEMBERS OF THE TRANSMEMBRANE-4 SUPERFAMILY, WITH DISTINCT SINGLE TRANSMEMBRANE-DOMAINED PROTEINSIN DIFFERENTIATED UROTHELIAL CELLS

The transmembrane 4 (TM4) superfamily contains many important leukocyt e differentiation-related surface proteins including CD9, CD37, CD53, and CD81; tumor-associated antigens including CD63/ME491, CO029, and S AS; and a newly identified metastasis suppressor gene R2. Relatively l ittle is known, however, about the structure and aggregation state of these four transmembrane-domained proteins. The asymmetrical unit memb rane (AUM), believed to play a major role in stabilizing the apical su rface 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 providin g unique opportunities for studying membrane structure and function. T o better understand how these proteins interact to form the 16-nm part icles, we analyzed their nearest neighbor relationship by chemical cro ss-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 compl exes that are stable in SDS, and that UPII can be readily crosslinked to form homodimers. The formation of UPII homodimers is sensitive, how ever, to octyl glucoside that can solubilize the AUMs. These data sugg est that there exist two types of 16-nm AUM particles that contain UPI a/UPII or UPIb/UPIII, and support a model in which the UPIa and UPII o ccupy the inner and outer domains, respectively, of the UPIa/UPII part icle. This model can account for the apparent ''redundancy'' of the ur oplakins, as the structurally related UPIa and UPlb, by interacting wi th different partners, may play different roles in AUM formation. The model also suggests that AUM plaques with different uroplakin composit ions may differ in their assembly, and in their abilities to interact with an underlying cytoskeleton. Our data indicate that two closely re lated TM4 proteins, UPIa and UPIb, can be present in the same cell, in teracting with distinct partners. AUM thus provides an excellent model system for studying the targeting, processing, and assembly of TM4 pr oteins.