FORMATION OF ASYMMETRIC UNIT MEMBRANE DURING UROTHELIAL DIFFERENTIATION

Mammalian urothelium undergoes unique membrane specialization during t erminal differentiation making numerous rigid-looking membrane plaques (0.3-0.5 mu m diameter) that cover the apical cell surface. The outer leaflet of these membrane plaques is almost twice as thick as the inn er leaflet hence the name asymmetric unit membrane (AUM). Ultrastructu ral studies established that the outer leaflet of AUM is composed of 1 6 nm particles forming two dimensional crystals, and that each particl e forms a 'twisted ribbon' structure. We showed recently that highly p urified bovine AUMs contain four major integral membrane proteins: uro plakins Ia (27 kD), Tt, (28 kD), II (15 kD) and III (47 kD). Studies o f the protease sensitivity of the different subdomains of uroplakins a nd other considerations suggest that UPIa and UPIb have 4 transmembran e domains, while UPII and UPIII have only one transmembrane domain. Ch emical crosslinking studies showed that UPIa and UPIb, which share 39% amino acid sequence, are topologically adjacent to UPII and UPIII, re spectively, thus raising the possibility that there exist two biochemi cally distinct AUM particles, i.e., those containing UPIa/UPII vs. UPI b/UPIII. Bovine urothelial cells grown in the presence of 3T3 feeder c ells undergo clonal growth forming stratified colonies capable of synt hesizing and processing all known uroplakins. Transgenic mouse studies showed that a 3.6 kb 5'-flanking sequence of mouse uroplakin II gene can drive the expression of bacterial LacZ gene to express in the urot helium. Further studies on the biosynthesis, assembly and targeting of uroplakins will offer unique opportunities for better understanding t he structure and function of AUM as well as the biology of mammalian u rothelium.