Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5-E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.cell signaling ͉ endocytosis ͉ gene targeting

Polycystin-1 (PC-1) is the product of the PKD1 gene, which is mutated in autosomal dominant polycystic kidney disease. We show that the Na,K-ATPase alpha-subunit interacts in vitro and in vivo with the final 200 amino acids of the polycystin-1 protein, which constitute its cytoplasmic C-terminal tail. Functional studies suggest that this association may play a role in the regulation of the Na,K-ATPase activity. Chinese hamster ovary cells stably expressing the entire PC-1 protein exhibit a dramatic increase in Na,K-ATPase activity, although the kinetic properties of the enzyme remain unchanged. These data indicate that polycystin-1 may contribute to the regulation of Na,K-ATPase activity in kidneys in situ, thus modulating renal tubular fluid and electrolyte transport.

The sorting and regulation of the Na,K and H,K-ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery. The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated. We have begun a series of yeast two-hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.

The short isoform of the Na'lCa'' exchanger (67 kDa) that is produced by alternative splicing during the expression of the 6 kb canine exchanger cDNA in 293 cells was separately expressed in the same system. The protein consisted of the five N-terminal transmembrane segments and of a large portion of the main hydrophilic loop, but lacked the six C-terminal hydrophobic segments of the regular protein (108 kDa). Very high RNA levels were found after transient cell transfection with plasmid DNA encoding this truncated isoform. The RNA processing, the translation and targeting of the resulting protein to th; plasma membrane appeared to be less efficient than those of the 108-kDa polypeptide produced in the same system. The Na+-dependent Ca*+-uptake activity of 293 cells expressing the short isoform was measured by an isotopic rapid filtration method, whereas the current associated with Caz' extrusion was measured in electrophysiological patch-clamp experiments. The results showed that the expressed isoform functioned in the typical reverse and forward Na+/Caz+ exchange modes. In both the electrophysiological and the isotopic measurements the activity of the short isoform was 6-7-fold lower than that of the 108-kDa protein expressed in the same system. However, lower amounts of the short isoform reached the plasma membrane : its specific activity could thus be significantly higher. Possibly, the short isoform could form a dimer in which a second 67 kDa polypeptide replaces the C-terminal part of the 108-kDa protein.