Multiple types of degradative enzymes, including cathepsins of the cysteine protease family, have been implicated in the regulation of angiogenesis and invasion during cancer progression. Several cysteine cathepsins are up-regulated in a mouse model of pancreatic islet cell carcinogenesis (RIP1-Tag2), and tumor progression is impaired following their collective pharmacologic inhibition. Using null mutations of four of the implicated cysteine cathepsins, we have now dissected their individual roles in cancer development. Mutants of cathepsins B or S impaired tumor formation and angiogenesis, while cathepsin B or L knockouts retarded cell proliferation and tumor growth. Absence of any one of these three genes impaired tumor invasion. In contrast, removal of cathepsin C had no effect on either tumor formation or progression. We have identified E-cadherin as a target substrate of cathepsins B, L, and S, but not cathepsin C, potentially explaining their differential effects on tumor invasion. Furthermore, we detected analogous increases in cathepsin expression in human pancreatic endocrine neoplasms, and a significant association between increased levels of cathepsins B and L and tumor malignancy. Thus individual cysteine cathepsin genes make distinctive contributions to tumorigenesis.[Keywords: Cancer; mouse models; proteases; cysteine cathepsins; tumor microenvironment; pancreatic endocrine cancer] Supplemental material is available at http://www.genesdev.org.

Chemokine receptors serve as portals of entry for certain intracellular pathogens, most notably human immunodeficiency virus (HIV). Myxoma virus is a member of the poxvirus family that induces a lethal systemic disease in rabbits, but no poxvirus receptor has ever been defined. Rodent fibroblasts (3T3) that cannot be infected with myxoma virus could be made fully permissive for myxoma virus infection by expression of any one of several human chemokine receptors, including CCR1, CCR5, and CXCR4. Conversely, infection of 3T3-CCR5 cells can be inhibited by RANTES, anti-CCR5 polyclonal antibody, or herbimycin A but not by monoclonal antibodies that block HIV-1 infection or by pertussis toxin. These findings suggest that poxviruses, like HIV, are able to use chemokine receptors to infect specific cell subtypes, notably migratory leukocytes, but that their mechanisms of receptor interactions are distinct.

ROMK channels are responsible for K؉ secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH i ) with acidification causing channel closure (effective pK a ϳ6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP 2 ) is critical for opening of the inwardly rectifying K ؉ channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP 2 and by pH i . We find that disruption of PIP 2 -ROMK1 interaction not only decreases single-channel open probability (P o ) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK a shifted to pH ϳ7.8), such that the subconductance channels are relatively quiescent at physiological pH i . Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P o for the subconductance channels by alkalinization is not associated with an increase in PIP 2 -channel interaction. Thus, direct interaction with PIP 2 is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH i sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH i occurs via a mechanism distinct from the regulation by PIP 2 .Potassium channels play important roles in the regulation of potassium transport in kidney (1). Recently, cDNAs for the renal K ϩ channels and splice isoforms, ROMK1, -2, and -3, have been isolated (2-4). ROMKs belong to a large family of inward rectifier K ϩ channels, which also includes the strongly rectifying IRK1, the G protein-gated GIRK1, and the pancreatic ␤-cell inward rectifier (5). These cDNAs encode polypeptides of ϳ300 -500 amino acids, which share ϳ40% or more amino acid identity and have the common structure of a cytoplasmic N terminus, two hydrophobic segments that span the membrane as ␣-helices, one pore-forming partial membranespanning region, and a long cytoplasmic C terminus.Opening of the G protein-gated GIRK channels requires G protein ␤␥ subunits. Other inward rectifier K ϩ channels, such as ROMK1 and IRK1, are constitutively open (5). Inward rectifier K ϩ channels run down (close) when inside-out membrane patches are excised into ATP-free, Mg 2ϩ -containing solution. We and others (6 -9) recently found that direct interaction of inward rectifier channels with the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 is critical for channel opening. Reduction of membrane PIP 2 via activation of the Mg 2ϩ -dependent lipid phosphatases causes channel run-down. Direct application of PIP 2 -containing liposomes to membrane patches reactivates run-down channels (6 -9). Furthermore, PIP 2 is important for regulation of the G protein-gated channels by G␤␥ and by intracellular Na ϩ (8 -11) and modulates ATP sensitivity of K ATP channels (12, 13).ROMK1 channels are also regulated by cAMP-dependent protein kina...

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ROMK channels are present in the cortical collecting ducts of kidney and are responsible for K(+) secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K(+) secretion in cortical collecting ducts. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed in Xenopus laevis oocytes and cultured Madin-Darby canine kidney cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ~6 h. Coexpression with the Lys44-->Ala dominant-negative mutant dynamin, but not wild-type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of the Lys44-->Ala dynamin mutant on endocytosis of the channel. Coimmunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in Madin-Darby canine kidney cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca 2؉ -permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca 2؉ -permeable nonselective cation currents. Co-expression of a loss-offunction mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking ϳ2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.Autosomal dominant (AD) polycystic kidney disease (PKD) 1 is characterized by progressive enlargement of fluid-filled cysts in kidney and other tissues such as liver and pancreas, leading to loss-of-function in the kidneys and occasional mass effects in the liver (1). ADPKD is caused by mutations in one of the two genes, PKD1 and PKD2, which are responsible for ϳ85 and ϳ15% of cases, respectively (2, 3). Elucidation of function of polycystin-1 and polycystin-2, encoded by PKD1 and PKD2, respectively, is critical for understanding how mutations in these genes cause cyst formation.Polycystin-1 is a large protein consisting of 4,302 amino acids (4). The predicted structure of polycystin-1 includes a large N-terminal extracellular region (ϳ3,109 amino acids), eleven predicted transmembrane (TM) domains (ϳ993 amino acids), and a small C-terminal cytoplasmic tail (ϳ200 amino acids) (4, 5). The N-terminal extracellular portion contains two leucinerich repeats, a C-type lectin domain, 16 copies of unique immunoglobulin (Ig)-like PKD domains, an LDL-A-related motif, and a region of homology with a sea urchin receptor for egg jelly (suREJ) (5). The extracellular leucine-rich repeat, C-type lectin, Ig-like PKD domains, and LDL-A-related motif are potential sites for protein-protein and protein-carbohydrate interactions (5). The area of homology of polycystin-1 with the suREJ protein extends over ϳ1,000 amino acids from the last Ig-like PKD domain to the first TM domain (5). The suREJ protein is located on the sperm head and is involved in the influx of Ca 2ϩ ions from the extracellular space and triggering of the acrosome reaction (6).Polycystin-2 is a 968 amino acid protein with six predicted membrane-spanning domains. The region of six TM domains of polycystin-2 has significant sequence homology with the voltage-gated Ca 2ϩ and Na ϩ channels, and transient receptor potential (TRP) channels (3, 5, 7). The TM region of polycystin-2 also share ϳ50% sequence homology with the last 6 TM domains ...

affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cellattached recordings, application of PIP 2 to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP 2 content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP 2 content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K ؉ secretion in CCD by hormones that activate PKC.

Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for human immunodeficiency virus (HIV) and the poxvirus, myxoma virus, involve appropriating chemokine receptors.In this report we demonstrate that activation of multiple intracellular tyrosine phosphorylation events rapidly ensues following virus adsorption to NIH 3T3.CD4.CCR5 cells and affects the ultimate level of myxoma virus replication. UV-inactivated myxoma virus induces the rapid phosphorylation of CCR5 on tyrosine residues, the association of CCR5 with Jaks and p56 lck , and their phosphorylation-activation within minutes of virus adsorption. Additionally, we provide evidence for myxoma virus-inducible signal transducers and activators of transcription (Stat) and insulin receptor substrate (IRS) activation. In contrast to CCR5 activation effected by HIV Env protein, these myxoma virusinducible phosphorylation events are not sensitive to pertussis toxin treatment. Moreover, in cells that are non-permissive for myxoma virus infection, we provide evidence that myxoma virus fails to invoke this tyrosine phosphorylation cascade. Consistent with the observation that infection of CCR5-expressing cells is blocked by herbimycin A and the Jak 2 inhibitor, tyrophostin AG490, we infer that viral infectivity may be dependent on non-G-protein-coupled signal transduction pathways triggered by the infecting myxoma virus particle. This provides a novel post-binding mechanism by which viruses can co-opt a cellular receptor to permit productive virus infection.Poxviruses are DNA viruses that replicate autonomously in the cytoplasm of infected cells. They have been the subject of intensive study, based largely on their severe pathogenesis in humans and a variety of domestic animals (1). Until its global irradication, smallpox, caused by an orthopoxvirus, was one of the most serious diseases of mankind. Myxoma virus, a member of the Leporipoxvirus genus, is the causative agent of myxomatosis, a lethal disease of the European rabbit (2). Myxomatosis is characterized by extensive fulminating lesions and severe immune dysfunction accompanied by supervening Gram-negative bacterial infections of the respiratory tract (3).Recently, evidence was provided that myxoma virus may utilize chemokine receptors to initiate infection (4). Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for HIV 1 and myxoma virus, includes appropriating chemokine receptors (4 -6). Chemokines and their receptors are critical for the clearance of infectious pathogens. Specifically, chemokines are implicated in directing lymphocyte trafficking to sites of infection and in activating the effector functions of these immune cells to eliminate infectious pathogens (7). Thus, viral subversion of chemokine receptors is an effective way to modulate chemokine-receptor-mediated interactions that would invoke an immune response against the invading virus. Indeed, herpesviruses and poxviruses subvert a host immune response ...