Organization of uroplakin subunits: transmembrane topology, pair formationand plaque composition

The apical surfaces of urothelial cells are almost entirely covered with pl aques consisting of crystalline, hexagonal arrays of 16 nm uroplakin partic les. Although all four uroplakins, when SDS-denatured, can be digested by c hymotrypsin, most uroplakin domains in native urothelial plaques are resist ant to the enzyme. suggesting a tightly packed structure. The only exceptio n is the C-terminal, cytoplasmic tail of UPIII (UPIII) which is highly susc eptible to proteolysis, suggesting a loose configuration. When uroplakins a re solubilized with 2 degrees (degrees) octylglucoside and fractionated wit h ion exchangers, UPIa and UPII were bound as a complex by a cation exchang er. whereas UPIb and UPIII were bound by an anion exchanger. This result is consistent with the fact that UPIa and UPIb are cross-linked to UPII and U PIII, respectively, and suggests that the four uroplakins form two pairs co nsisting of UPIa/II and UPIb/III. Immunogold labelling using a new mouse mo noclonal antibody, AU1, revealed that UPIII is present in all urothelial pl aques. indicating that the two uroplakin pairs are not segregated into two different types of urothelial plaque and that all plaques must have a simil ar uroplakin composition. Taken together, these results indicate that uropl akins form a tightly packed structure, that the four uroplakins interact sp ecifically forming two pairs, and that both uroplakin pairs are required fo r normal urothelial plaque formation.