Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

Sensitivity of blood pressure to dietary salt is a common feature in subjects with hypertension. These features are exemplified by the mendelian disorder, Liddle's syndrome, previously shown to arise from constitutive activation of the renal epithelial sodium channel due to mutation in the beta subunit of this channel. We now demonstrate that this disease can also result from a mutation truncating the carboxy terminus of the gamma subunit of this channel; this truncated subunit also activates channel activity. These findings demonstrate genetic heterogeneity of Liddle's syndrome, indicate independent roles of beta and gamma subunits in the negative regulation of channel activity, and identify a new gene in which mutation causes a salt-sensitive form of human hypertension.

Autosomal recessive pseudohypoaldosteronism type I is a rare life-threatening disease characterized by severe neonatal salt wasting, hyperkalaemia, metabolic acidosis, and unresponsiveness to mineralocorticoid hormones. Investigation of affected offspring of consanguineous union reveals mutations in either the alpha or beta subunits of the amiloride-sensitive epithelial sodium channel in five kindreds. These mutations are homozygous in affected subjects, co-segregate with the disease, and introduce frameshift, premature termination or missense mutations that result in loss of channel activity. These findings demonstrate the molecular basis and explain the pathophysiology of this disease.

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Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the 13 or y subunit of the epithelial Na channel. All reported

Activity of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis and blood pressure. Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or alter PY domains in the carboxyl termini of ␤ or ␥ ENaC subunits, leading to increased channel activity. In this study we investigated the mechanism of this effect by analysis of wild-type and mutant ENaC activity in Xenopus oocytes. By inhibiting insertion of new channels into the plasma membrane with brefeldin A, we demonstrate that the half-life of the activity of channels containing Liddle's mutations is markedly prolonged compared with wildtype channels (t1 ⁄2 of 30 h in mutant versus 3.6 in wildtype, p < 0.001). We investigated the involvement of clathrin-coated pit-mediated endocytosis by co-expressing a dominant-negative dynamin mutant with wildtype ENaC in oocytes. Expression of this specific inhibitor of endocytosis leads to a large increase in the activity of wild-type channels, demonstrating that normal turnover of this channel is through the clathrincoated pit pathway. In contrast, co-expression of Liddle's mutations and dynamin mutants leads to no further increase in channel activity, consistent with one of the effects of Liddle's mutations being the loss of endocytosis of these channels. These findings demonstrate the normal mechanism of turnover of ENaC from the cell surface and demonstrate a mechanism that can account for the increased number of channels in the plasma membrane seen in Liddle's syndrome.Expression of the amiloride-sensitive epithelial sodium channel (ENaC) 1 in the plasma membrane determines the sodium permeability of many epithelia involved in sodium reabsorption. The channel is regulated by hormones such as aldosterone, insulin, and antidiuretic hormone that increase its activity by diverse mechanisms including increase in channel synthesis, incorporation of channels from an intracellular pool to the plasma membrane, and induction of post-translational modifications of the channel proteins (1). There are fewer known mechanisms that decrease sodium permeability in cells expressing ENaC. Recently, there have been several studies that implicate the carboxyl terminus of the channel subunits in processes that normally down-regulate its activity. Mutations in the human genes of the ␤ and ␥ subunits cause a form of salt-sensitive hypertension known as Liddle's syndrome (2, 3). These mutations introduce frameshifts or premature stop codons that delete the intracellular carboxyl-terminal domains of the ␤ or ␥ subunits, or they substitute residues in a prolinerich motif present in the carboxyl termini of both subunits (4, 5). Expression of ENaC channels with truncated ␤ or ␥ subunits in Xenopus oocytes induces amiloride-sensitive whole-cell currents 3-5-fold larger in magnitude than wild-type channels (6). Two mechanisms have been proposed to explain the increase in sodium permeability induced by Liddle's mutations: an increase in the number of channels expressed at the cell surface, a...