A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.

The clinical impact of the fibrate and thiazolidinedione drugs on dyslipidemia and diabetes is driven mainly through activation of two transcription factors, peroxisome proliferator-activated receptors (PPAR)-α and PPAR-γ. However, substantial differences exist in the therapeutic and side-effect profiles of specific drugs. This has been attributed primarily to the complexity of drug-target complexes that involve many coregulatory proteins in the context of specific target gene promoters. Recent data have revealed that some PPAR ligands interact with other non-PPAR targets. Here we review concepts used to develop new agents that preferentially modulate transcriptional complex assembly, target more than one PPAR receptor simultaneously, or act as partial agonists. We highlight newly described on-target mechanisms of PPAR regulation including phosphorylation and nongenomic regulation. We briefly describe the recently discovered non-PPAR protein targets of thiazolidinediones, mitoNEET, and mTOT. Finally, we summarize the contributions of on- and off-target actions to select therapeutic and side effects of PPAR ligands including insulin sensitivity, cardiovascular actions, inflammation, and carcinogenicity.

Protein tyrosine phosphatase RQ (PTPRQ) was initially identified as a protein tyrosine phosphatase (PTPase)-like protein that is upregulated in a model of renal injury. Here we present evidence that, like PTEN, the biologically important enzymatic activity of PTPRQ is as a phosphatidylinositol phosphatase (PIPase). The PIPase specificity of PTPRQ is broader than that of PTEN and depends on different amino acid residues in the catalytic domain. In vitro, the recombinant catalytic domain of PTPRQ has low PTPase activity against tyrosine-phosphorylated peptide and protein substrates but can dephosphorylate a broad range of phosphatidylinositol phosphates, including phosphatidylinositol 3,4,5-trisphosphate and most phosphatidylinositol monophosphates and diphosphates. Phosphate can be hydrolyzed from the D3 and D5 positions in the inositol ring. PTPRQ does not have either of the basic amino acids in the catalytic domain that are important for the PIPase activity of PTEN or the sequence motifs that are characteristic of type II phosphatidylinositol 5-phosphatases. Instead, the PIPase activity depends on the WPE sequence present in the catalytic cleft of PTPRQ, and in the ''inactive'' D2 domains of many dual-domain PTPases, in place of the WPD motif present in standard active PTPases. Overexpression of PTPRQ in cultured cells inhibits proliferation and induces apoptosis. An E2171D mutation that retains or increases PTPase activity but eliminates PIPase activity, eliminates the inhibitory effects on proliferation and apoptosis. These results indicate that PTPRQ represents a subtype of the PTPases whose biological activities result from its PIPase activity rather than its PTPase activity.

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoRdissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.Nuclear receptors are a family of transcriptional regulators whose activity can be modulated by their binding to small molecule compounds, such as hormones and metabolites. For many members of the family, this property has allowed their use as drug targets (1). In most cases, however, full activation or inhibition of the receptor is not desired. Instead, agonists are required that only partially activate the receptor. Partial agonists can display tissue-specific activation or repression of nuclear receptors, as has been shown for the estrogen receptor partial agonist raloxifen (2). However, little is known about the molecular mechanisms leading to partial versus full agonism.Another example for nuclear receptors, which, if they are to be used as drug targets, require partial agonists, are the liver X receptors ␣ (LXR␣, 2 NR1H3) and ␤ (LXR␤, NR1H2). They have been shown to play a central role in the transcriptional regulation of lipid and cholesterol homeostasis and inflammation (3-7). Activation of LXR-dependent transcription leads to increased expression of cholesterol transporters (8 -11) and has been shown to enhance the efflux of cholesterol from macrophages (10 -12), reducing the formation of atherosclerotic plaques (13)(14)(15). This observation has raised hopes that the manipulation of LXR activity would be of therapeutic value in the treatment of lipid disorders and atherosclerosis. However, activation of LXRs by agonistic compounds induces the expression of enzymes involved in the synthesis of fatty acids in liver cells (16 -19). As a consequence, agonists for LXRs cause liver steatosis and elevated serum triglyceride levels in mice (19,20). Thus, to develop LXR ligands as drugs for the treatment of atherosclerosis, partial, selective activator...

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43-1 and dna52-1 cells undergo cell-cycle arrest at the restrictive temperature (37 degrees C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna52-1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.

Various insults cause ototoxicity in mammals by increasing oxidative stress leading to apoptosis of auditory hair cells (HCs). The thiazolidinediones (TZDs; e.g., pioglitazone) and fibrate (e.g., fenofibrate) drugs are used for the treatment of diabetes and dyslipidemia. These agents target the peroxisome proliferator-activated receptors, PPARγ and PPARα, which are transcription factors that influence glucose and lipid metabolism, inflammation, and organ protection. In this study, we explored the effects of pioglitazone and other PPAR agonists to prevent gentamicin-induced oxidative stress and apoptosis in mouse organ of Corti (OC) explants. Western blots showed high levels of PPARγ and PPARα proteins in mouse OC lysates. Immunofluorescence assays indicated that PPARγ and PPARα proteins are present in auditory HCs and other cell types in the mouse cochlea. Gentamicin treatment induced production of reactive oxygen species (ROS), lipid peroxidation, caspase activation, PARP-1 cleavage, and HC apoptosis in cultured OCs. Pioglitazone mediated its anti-apoptotic effects by opposing the increase in ROS induced by gentamicin, which inhibited the subsequent formation of 4-hydroxy-2-nonenal (4-HNE) and activation of pro-apoptotic mediators. Pioglitazone mediated its effects by upregulating genes that control ROS production and detoxification pathways leading to restoration of the reduced:oxidized glutathione ratio. Structurally diverse PPAR agonists were protective of HCs. Pioglitazone (PPARγ-specific), tesaglitazar (PPARγ/α-specific), and fenofibric acid (PPARα-specific) all provided >90% protection from gentamicin toxicity by regulation of overlapping subsets of genes controlling ROS detoxification. This study revealed that PPARs play important roles in the cochlea, and that PPAR-targeting drugs possess therapeutic potential as treatment for hearing loss.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR␥ upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR␥ is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR␥ signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR␥ and enhanced nuclear translocation and ligand-independent transcription of PPAR␥ target genes. In contrast, Cav1 overexpression sequestered PPAR␥ in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR␥'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the liganddependent transcriptional activity of PPAR␥ and to inhibit cell proliferation. Ligand-activated PPAR␥ also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR␥ regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR␥ to ligands, limiting proliferation of gastric epithelial cells.Peroxisome proliferator-activated receptor ␥ (PPAR␥) belongs to the nuclear receptor (NR) superfamily (31). Infection by Helicobacter pylori is a major risk factor for gastric cancer (GC) in humans (68). H. pylori increases the expression of PPAR␥, cytokines, and eicosanoids, while PPAR␥ protects the gastric epithelium by inhibiting apoptosis of host cells (19) and inflammation (42). PPAR␥ ligands (glitazones; 15-deoxy-prostaglandin J 2 ) have been shown to inhibit proliferation and induce growth arrest or apoptosis in human GC cell lines (32,52,53). PPAR␥ knockout (KO) mice are susceptible to chemically induced gastric carcinogenesis (36). In humans, the common "partial loss of function" gene polymorphism (Pro12Ala) is correlated with an increased risk of GC, suggesting a role for PPAR␥ as a tumor suppressor in the stomach (67).PPAR␥ inhibits cell proliferation by several mechanisms, including inhibition of cyclin D1 expression, promotion of its proteasomal degradation, and upregulation of cyclin-dependent kinase (CDK) inhibitors (20,55,65). Members of the Ras/mitogen-activated protein kinase (MAPK) cascade, such as extracellular signal-regulated kinases 1/2 (ERK1/2), counteract this effect by inducing cyclin D1 expression and reducing PPAR␥ activity by phosphorylation on serine 84 (serine 82 in mouse) in its N-terminal activation function (AF1) (7). Cav1, a scaffold protein of plasma membrane caveolae (46), attenuates ERK1/2 activation and cell growth by sequestration of upstream MAPK cascade components, including growth factor receptors, Ras, Raf, and MEK1. In contrast, Cav1...

The finding that rosiglitazone may increase risk for cardiovascular events has led to regulatory guidelines requiring demonstration of cardiovascular safety in appropriate outcome trials for new type 2 diabetes mellitus drugs. The emerging data on the possibly increased risk of bladder cancer with pioglitazone may prompt the need for post-approval safety studies for new drugs. Since PPAR-α and -γ affect key cardiometabolic risk factors (diabetic dyslipidemia, insulin resistance, hyperglycemia, and inflammation) in a complementary fashion, combining their benefits has emerged as a particularly attractive option. New PPAR-targeted therapies that balance the relative potency and/or activity toward PPAR-α and -γ have shown promise in retaining efficacy while reducing potential side effects.