Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B

Processing endopeptidase deficiency in neurohypophysial secretory granules of the diabetes insipidus (Brattleboro) rat

The homozygote Brattleboro rat exhibits a hereditary diabetes insipidus due to a deficiency of vasopressin, the antidiuretic hormone. It has previously been shown that in this animal a single nucleotide deletion in the provasopressin gene leads to a mutant precursor with a C-terminal amino acid sequence different from that of the wild-type. However the N-terminal region including the hormone moiety, the processing signal as well as the first two-thirds of the neurophysin is entirely preserved and absence of maturation has to be explained by an additional cause. We show here that the neurohypophysis of the homozygote Brattleboro rat, in contrast to the adenohypophysis, displays a significant decrease in the Lys-Arg processing endopeptidase activity when compared to the heterozygote or the wild-type Wistar. It is suggested that hypothalamic vasopressinergic neurons of the homozygote Brattleboro rat display a deficiency in the processing enzyme in contrast to the oxytocinergic neurons in which processing of prooxytocin is normal

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E(2) (PGE(2)) with at least a 1,000 fold selectivity for COX-2 (IC(50)=41±14 nM) over COX-1 (IC(50)>50 μM). Indomethacin was a potent inhibitor of both COX-1 (IC(50)=18±3 nM) and COX-2 (IC(50)=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B(2) (TXB(2)) by Ca(2+) ionophore-challenged human platelets (IC(50)>50 μM and 4.1±1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a K(i) value of 140±68 μM for the initial reversible binding to enzyme and a k(2) value of 0.11±0.06 s(−1) for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s(−1), respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t(1/2)=1–3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC(50)=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE(2) production (COX-2) in a human whole blood assay with a potency (IC(50)=0.28±0.04 μM) similar to indomethacin (IC(50)=0.68±0.17 μM). In contrast, DFU was at least 500 times less potent (IC(50)>97 μM) than indomethacin at inhibiting coagulation-induced TXB(2) production (COX-1) (IC(50)=0.19±0.02 μM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 μM), DFU inhibited COX-1 with an IC(50) value of 13±2 μM as compared to 20±1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac>indomethacin∼naproxen>nimesulide∼ meloxicam∼piroxicam>NS-398∼SC-57666>SC-58125>CGP 28238∼etodolac>L-745,337>DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED(50) of 1.1 mg kg(−1) vs 2.0 mg kg(−1) for indomethacin) and hyperalgesia (ED(50) of 0.95 mg kg(−1) vs 1.5 mg kg(−1) for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED(50)=0.76 mg kg(−1) vs 1.1 mg kg(−1) for indomethacin). 8. In a sensitive model in which (51)Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg(−1), b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg(−1)), meloxicam (3 mg kg(−1)) or etodolac (10–30 mg kg(−1)). A 5 day administration of DFU in squirrel monkeys (100 mg kg(−1)) did not affect chromium leakage in contrast to diclofenac (1 mg kg(−1)) or naproxen (5 mg kg(−1)). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor