The human kidney is composed of roughly 1.2-million renal tubules that must maintain their tubular structure to function properly. In autosomal dominant polycystic kidney disease (ADPKD) cysts develop from renal tubules and enlarge independently, in a process that ultimately causes renal failure in 50% of affected individuals. Mutations in either PKD1 or PKD2 are associated with ADPKD but the function of these genes is unknown. PKD1 is thought to encode a membrane protein, polycystin-1, involved in cell-cell or cell-matrix interactions, whereas the PKD2 gene product, polycystin-2, is thought to be a channel protein. Here we show that polycystin-1 and -2 interact to produce new calcium-permeable non-selective cation currents. Neither polycystin-1 nor -2 alone is capable of producing currents. Moreover, disease-associated mutant forms of either polycystin protein that are incapable of heterodimerization do not result in new channel activity. We also show that polycystin-2 is localized in the cell in the absence of polycystin-1, but is translocated to the plasma membrane in its presence. Thus, polycystin-1 and -2 co-assemble at the plasma membrane to produce a new channel and to regulate renal tubular morphology and function.

Angiogenesis, the formation of new microvasculature by capillary sprouting, is crucial for tumour development. Hypoxic regions of solid tumours produce the powerful and directly acting angiogenic protein VEGF/VPF (vascular endothelial growth factor/vascular permeability factor). We now investigate the signal transduction pathway involved in hypoxic induction of VEGF expression. Hypoxia is known to induce a tyrosine kinase cascade that results in the activation of nitrogen-fixation genes in Rhizobium meliloti, and activation of tyrosine kinases is critical in signalling triggered by growth factors and ultraviolet light. We show here that genistein, an inhibitor of protein tyrosine kinase, blocks VEGF induction. Hypoxia increases the kinase activity of pp60c-src and its phosphorylation on tyrosine 416 but does not activate Fyn or Yes. Expression of either a dominant-negative mutant form of c-Src or of Raf-1 markedly reduces VEGF induction. VEGF induction by hypoxia in c-src(-) cells is impaired, although there is a compensatory activation of Fyn. Our results provide an insight into hypoxia-triggered intracellular signalling, define VEGF as a new downstream target for c-SRC, and suggest a role for c-SRc in promoting angiogenesis.

PKD1 and PKD2 are two recently identified genes that are responsible for the vast majority of autosomal polycystic kidney disease, a common inherited disease that causes progressive renal failure. PKD1 encodes polycystin, a large glycoprotein that contains several extracellular motifs indicative of a role in cell-cell or cell-matrix interactions, and the PKD2 encodes a protein with homology to a voltageactivated calcium channel and to PKD1. It is currently unknown how mutations of either protein functionally cause autosomal polycystic kidney disease. We show that PKD1 and PKD2 interact through their C-terminal cytoplasmic tails. This interaction resulted in an up-regulation of PKD1 but not PKD2. Furthermore, the cytoplasmic tail of PKD2 but not PKD1 formed homodimers through a coiled-coil domain distinct from the region required for interaction with PKD1. These interactions suggest that PKD1 and PKD2 may function through a common signaling pathway that is necessary for normal tubulogenesis and that PKD1 may require the presence of PKD2 for stable expression.Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease that accounts for 8-10% of endstage renal disease. ADPKD is genetically heterogeneous with loci mapped to chromosome 16p13.3 (PKD1) (1) and to chromosome 4q21-23 (PKD2) (2-4), with the likelihood of a third unmapped locus. PKD1 (5, 6) and PKD2 (4) have recently been cloned and found to be broadly expressed (4, 7). The predicted PKD1 protein is a glycoprotein with multiple transmembrane domains and a C-terminal cytoplasmic tail of 225 amino acids. The N-terminal extracellular region of Ϸ2,557 amino acids contains multiple domains that implicate PKD1 in cell-cell or cell-matrix interactions. These include leucine-rich repeats, a C-type lectin domain, 16 immunoglobulin-like repeats, and 4 type III fibronectin-related domains. PKD2 encodes an integral membrane protein of 968 amino acids containing six transmembrane domains flanked by cytoplasmic N and C termini. Homology of PKD2 to the ␣ 1E-1 subunit of a voltage-activated calcium channel (VACC␣ 1E-1 ) (4) is evident throughout most of the transmembrane domains and the cytoplasmic C-terminal tail, including a potential E-F hand motif. Similarities between PKD1 and PKD2 are restricted to the transmembrane domains I through IV of PKD2. The predicted structures of PKD1 and PKD2, and their similar disease profiles, are highly suggestive of their involvement in a common signaling pathway that links extracellular adhesive events to alterations in ion transport (4).Although various functional abnormalities have been detected in cultured human epithelial cells isolated from cystic lesions of patients with ADPKD, these observations have not clarified the nature of the aberrant gene products caused by mutations of PKD1 and PKD2. Renal cysts are thought to arise through a process of persistent epithelial proliferation related to the lack of terminal differentiation. Both abnormal growth factor responsiveness (8-12) and the elevated e...

Signaling through the store-operated Ca 2+ release-activated Ca 2+ (CRAC) channel regulates critical cellular functions, including gene expression, cell growth and differentiation, and Ca 2+ homeostasis. Loss-of-function mutations in the CRAC channel pore-forming protein ORAI1 or the Ca 2+ sensing protein stromal interaction molecule 1 (STIM1) result in severe immune dysfunction and nonprogressive myopathy. Here, we identify gain-of-function mutations in the cytoplasmic domain of STIM1 (p.R304W) associated with thrombocytopenia, bleeding diathesis, miosis, and tubular myopathy in patients with Stormorken syndrome, and in ORAI1 (p.P245L), associated with a Stormorken-like syndrome of congenital miosis and tubular aggregate myopathy but without hematological abnormalities. Heterologous expression of STIM1 p.R304W results in constitutive activation of the CRAC channel in vitro, and spontaneous bleeding accompanied by reduced numbers of thrombocytes in zebrafish embryos, recapitulating key aspects of Stormorken syndrome. p.P245L in ORAI1 does not make a constitutively active CRAC channel, but suppresses the slow Ca 2+ -dependent inactivation of the CRAC channel, thus also functioning as a gain-of-function mutation. These data expand our understanding of the phenotypic spectrum of dysregulated CRAC channel signaling, advance our knowledge of the molecular function of the CRAC channel, and suggest new therapies aiming at attenuating store-operated Ca 2+ entry in the treatment of patients with Stormorken syndrome and related pathologic conditions. human genetics | calcium signaling C a 2+ influx in response to the depletion of intracellular Ca 2+ stores, or store-operated Ca 2+ entry, constitutes one of the major routes of Ca 2+ entry in all animal cells (1). Under physiological conditions, Ca 2+ influx is activated in response to numerous G protein-coupled receptors and receptor tyrosine kinases signaling via inositol-1,4,5-trisphosphate as a second messenger (2). Store-operated Ca 2+ entry is mediated primarily by the Ca 2+ release-activated Ca 2+ (CRAC) channel (3), which consists of the pore-forming subunits ORAI1-3 (or CRAC modulators 1-3) and Ca 2+ sensors, STIM1 and STIM2 (4-7). STIM proteins reside in the membrane of endoplasmic reticulum (ER), whereas ORAI proteins reside in the plasma membrane. STIM1 is a single transmembrane-spanning protein (8-12) that, in resting cells, exists as a dimer that binds Ca 2+ through two EF hand-containing domains located in the ER lumen (13). Depletion of Ca 2+ in the ER induces a series of molecular events in the conformation and localization of STIM1, initiated by the formation of higher-order oligomers, protein unfolding, and accumulation at discrete sites in the cell where the ER membrane is in close proximity to the plasma membrane (11,(13)(14)(15)(16). In these sites, STIM1 binds to the cytosolic C and N termini of ORAI1 (17, 18), resulting in channel activation and generation of a highly Ca 2+ -selective CRAC current, or I CRAC (3,19,20). I CRAC is responsible not only ...