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K Ling

Publications and source records attributed to K Ling.

14 recordsLinked to original sources
PubMed2001

Direct binding of beta-arrestins to two distinct intracellular domains of the delta opioid receptor.

beta-Arrestins regulate opioid receptor-mediated signal transduction and play an important role in opiate-induced analgesia and tolerance/dependence. This study was carried out to measure the direct interaction between beta-arrestins and opioid receptor. Immunoprecipitation experiments demonstrated that beta-arrestin 1 physically interacts with delta opioid receptor (DOR) co-expressed in human embryonic kidney 293 cells in an agonist-enhanced manner and truncation of the carboxyl terminus of DOR partially impairs the interaction. In vitro data from glutathione-S-transferase pull-down assay showed that the carboxyl terminus (CT) and the third intracellular loop (I3L) of DOR are both capable of and either domain is sufficient for binding to beta-arrestin 1 and 2. Surface plasmon resonance determination further revealed that binding of CT and I3L of DOR to beta-arrestin is additive, suggesting these two domains bind at distinctly different sites on beta-arrestin without considerable spatial hindrance. This study demonstrated for the first time the direct binding of beta-arrestins to the two distinct domains, the carboxyl terminus and the third intracellular loop, of DOR.

Amino Acid Sequence↗
PubMed2000

Identification of a human brain-specific isoform of mammalian STE20-like kinase 3 that is regulated by cAMP-dependent protein kinase.

A novel isoform of mammalian STE20-like kinase 3 (MST3) with a different 5' coding region from MST3, termed MST3b, was identified by searching through expressed sequence tag data base and obtained by rapid amplification of cDNA 5'-ends. MST3b was assigned to the long arm of human chromosome 13, D13S159-D13S280, by use of the National Center for Biotechnology Information sequence-tagged sites data base. Reverse transcription-polymerase chain reaction and Northern blot analysis with a probe derived from 5' distinct sequence of MST3b revealed that the expression of MST3b mRNA is restricted to the brain, in contrast to ubiquitous distribution of MST3 transcript. Western analysis confirmed the brain-specific expression of MST3b protein. In situ hybridization of rat brain sections with a MST3b-specific probe indicated that MST3b is widely expressed in different brain regions, with especially high expression in hippocampus and cerebral cortex. When expressed in human embryonic kidney 293 (HEK293) cells, MST3b effectively phosphorylated myelin basic protein, as well as undergoing autophosphorylation. Interestingly, expression of MST3, but not MST3b, in HEK293 cells was able to activate the endogenous p42/44 mitogen-activated protein kinase (MAPK) up to 4-fold, whereas neither isoform activated p38 MAPK under the same conditions. Further experiments demonstrated that MST3b, but not MST3, was effectively phosphorylated by activation of cyclic AMP-dependent protein kinase (PKA) in both in vivo and in vitro assays. The mutation of Thr-18 into Ala in MST3b (T18A), a putative PKA phosphorylation site that is absent in MST3, abolished its phosphorylation by PKA. Consequently, expression of the T18A mutant in HEK293 cells led to partial activation of p42/44 MAPK, indicating that MST3b is under the regulation of PKA. Taken together, our data provide evidence that the two isoforms of STE20-like kinase 3 are differentially distributed and regulated.

Animals↗
PubMed1999

Five-transmembrane domains appear sufficient for a G protein-coupled receptor: functional five-transmembrane domain chemokine receptors.

The putative seven-transmembrane (TM) domains have been the structural hallmark for the superfamily of heterotrimeric G protein-coupled receptors (GPCRs) that regulate a variety of cellular functions by mediating a large number of extracellular signals. Five-TM GPCR mutants of chemokine receptor CCR5 and CXCR4, the N-terminal segment of which connected directly to TM3 as a result of a deletion of TM1-2 and the first intracellular and extracellular loops, have been obtained in this study. Laser confocal microscopy and flow cytometry analysis revealed that these five-TM mutant GPCRs were expressed stably on the cell surface after transfection into human embryonic kidney 293 cells. The five-TM CCR5 and CXCR4 functioned as normal chemokine receptors in mediating chemokine-stimulated chemotaxis, Ca2+ influx, and activation of pertussis toxin-sensitive G proteins. Like the wild-type GPCRs, the five-TM mutant receptors also underwent agonist-dependent internalization and desensitization and were subjected to regulation by GPCR kinases and arrestins. Our study indicates that five-TM domains, at least in the case of CCR5 and CXCR4, appear to meet the minimum structural requirements for a functional GPCR and suggests possible existence of functional five-TM GPCRs in nature during evolution.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Anti-HIV agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and G protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors.

Trichosanthin (TCS), an active protein component isolated from a traditional Chinese medicinal herb Trichosanthes kirilowii, has been shown to inhibit HIV infection and has been applied in clinical treatment of AIDS. The recent development that chemokines and chemokine receptors play important roles in HIV infection led us to investigate the possible functional interaction of TCS with chemokines and their receptors. This study demonstrated that TCS greatly enhanced both RANTES (regulated upon activation, normal T cell expressed and secreted)- and stromal cell-derived factor (SDF)-1 alpha-stimulated chemotaxis (EC50 approximately equal to 1 nM) in leukocytes (THP-1, Jurkat, and peripheral blood lymphocyte cells) and activation of pertussis toxin-sensitive G proteins (EC50 approximately equal to 20 nM). TCS also significantly augmented chemokine-stimulated activation of chemokine receptors CCR5 and CXCR4 as well as CCR1, CCR2B, CCR3, and CCR4 transiently expressed in HEK293 cells. A mutant TCS with 4,000-fold lower ribosome-inactivating activity showed similar augmentation activity as wild-type TCS. Moreover, flow cytometry demonstrated that the specific association of TCS to the cell membranes required the presence of chemokine receptors, and laser confocal microscopy reveals that TCS was colocalized with chemokine receptors on the membranes. The results from TCS-Sepharose pull-down and TCS and chemokine receptor coimmunoprecipitation and cross-linking experiments demonstrated association of TCS with CCR5. Thus, our data clearly demonstrated that TCS synergizes activities of chemokines to stimulate chemotaxis and G protein activation, and the effects of TCS are likely to be mediated through its interaction with chemokine receptors.

Animals↗
PubMed1998

Differential efficacies of kappa agonists to induce homologous desensitization of human kappa opioid receptor.

The efficacies of different K agonists to activate K opioid receptor (kappaOR) and to induce acute homologous desensitization of kappaOR-mediated extracellular acidification response (ECAR) have been investigated using microphysiometry in Chinese hamster ovary (CHO) cells stably expressing kappaOR. Although efficacy of dynorphin A (DA) to stimulate kappaOR-mediated ECAR was comparable to that of U69593, DA displayed a significant higher efficacy to induce desensitization of kappaOR. Moreover, the half life for the recovery of kappaOR responsiveness after DA prechallenge was four times longer than those observed after U69593 or etorphine pretreatment. In contrast, the efficacy of DA to stimulate and desensitize mu opioid receptors (muOR) stably expressed in CHO cells were comparable to those of other mu agonists.

Animals↗
PubMed1998

Differential neurotoxicity of etorphine-like opiates: lack of correlation with their ability to activate opiate receptors.

The present study was undertaken to compare the neurotoxic effects of three etorphine-like opiates (etorphine, dihydroetorphine, and another derivative of oripavine) and heroin with their ability to activate opiate receptors in human neuroblastoma cell line SK-N-SH as well as in two other neuronal cell lines. Neurotoxicity was measured by using [3H]-thymidine incorporation analysis, cell viability measurement and Cytosensor microphysiometry. It was found that, in spite of the very similar molecular structures of these opiates, they displayed significant differences in cytotoxicity, with etorphine and another derivative of oripavine possessing high potency but dihydroetorphine and heroin little effect. However, neurotoxic potency of the opiates was not directly correlated to their ability to activate opioid receptors, as determined by [35S]-guanylyl-5'-O-(gamma-tho)-triphosphate binding assay. These findings provide clear evidence of differential neurotoxicity of etorphine-like opiates, and suggest that the neurotoxicity is not closely related to the molecular configuration required as opioid receptor agonist but is probably associated with the presence of a double bond in the structure.

Animals↗
PubMed1997

Nociceptin/orphanin FQ stimulates extracellular acidification and desensitization of the response involves protein kinase C.

A Chinese hamster ovary (CHO) cell line, CHO-ORL1, stably expressing human opioid receptor-like receptor 1 (ORL1) has been used to determine ORL1-mediated signaling events using microphysiometry. Nociceptin/orphanin FQ (N/OFQ), a specific endogenous agonist of ORL1, induced an increase in extracellular acidification rate (ECAR) in CHO-ORL1 cells. The ECAR response stimulated by N/OFQ was concentration-dependent and pertussis toxin-sensitive. Repeated exposures of the cells to N/OFQ caused desensitization of ORL1. The ECAR response was recovered at the half-life of approximately 12 min after the initial challenge. Pretreatment with inhibitor of cAMP-dependent kinase did not affect desensitization of ORL1. However, specific inhibitors for protein kinase C almost abolished N/OFQ-induced desensitization of extracellular acidification responsiveness, indicating the involvement of protein kinase C in the process.

Animals↗
PubMed1997

Molecular characterization and functional expression of opioid receptor-like1 receptor.

The opioid receptor-like1 receptor (ORL1), an orphan receptor whose human and murine complementary DNAs, has been characterized recently. ORL1 transcripts are particularly abundant in the central nervous system. We demonstrated that ORL1 expressed in human neuroblastoma SK-N-SH and SH-SY5Y cell lines by radioligand binding assay, reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis in the present study. Stimulation with ORL1 specific agonist, nociceptin/orphanin FQ, increased [35S]GTP gamma S binding to SK-N-SH cell membranes (EC50 = 14 +/- 0.45 nM), and attenuated forskolin-stimulated accumulation of cellular cAMP (EC50 = 0.80 +/- 0.45 nM), indicative that activation of ORL1 activates G proteins and inhibits adenylyl cyclase. Activation of ORL1 receptor was also accessed using CHO:hORL1 cell line by microphysiometer. Treatment of nociceptin/orphanin FQ increased extracellular acidification rate significantly.

Adenylyl Cyclase Inhibitors↗
PubMed1995

Differential response of the Ras exchange factor, Ras-GRF to tyrosine kinase and G protein mediated signals.

Ras-GRF, a guanine-nucleotide exchange factor that activates Ras p21, was tested for its ability to couple to either tyrosine kinase or heterotrimeric G protein signal transduction pathways. Ras-GRF failed to bind the SH2 and SH3 containing adaptor protein Grb2, either in vitro or in vivo. Furthermore, Ras-GRF did not form a stable complex with activated EGF receptor. However, as has been shown previously (Cen et al., 1994), the presence of Ras-GRF in NIH3T3 cells enhanced the activation of Ras induced by serum stimulation. A similar effect was not observed with PDGF stimulation. Moreover, serum stimulation lead to the hyperphosphorylation of Ras-GRF. Both the serum induced super-activation of Ras, and the hyperphosphorylation of Ras-GRF were blocked by pretreatment of cells with the Gi,o inhibitor pertussis toxin, but not by pretreatment with the tyrosine kinase inhibitor genistein. These results suggest that Ras-GRF has the capacity to mediate Ras activation initiated by signals using heterotrimeric G proteins.

3T3 Cells↗
PubMed1991

Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene.

We obtained transgenic tobacco plants expressing the papaya ringspot virus (PRV) coat protein (CP) gene by transformation via Agrobacterium tumefaciens. Expression was effectively monitored by enzyme-linked immunosorbent assays (ELISA) of crude tissue extracts. Subcloned plants derived from eight original Ro transformants were inoculated with potyviruses: tobacco etch (TEV), potato virus Y (PVY), and pepper mottle (PeMV). Plants that accumulated detectable levels of the PRV CP showed significant delay in symptom development and the symptoms were attenuated. Similar results were obtained with inoculated R1 plants. We conclude that the expression of the PRV CP-gene imparts protection against infection by a broad spectrum of potyviruses.

Base Sequence↗
PubMed1986

[Carcinoembryonic antigen and immunoglobin in gastric juice in the diagnosis of gastric cancer].

Levels of carcinoembryonic antigen (CEA) and immunoglobin (Ig) in gastric juice of 93 patients with benign and malignant gastric diseases were assayed. The CEA level in gastric cancer patients (55.73 +/- 38.26 ng/ml) was obviously higher than that in peptic ulcer (15.51 +/- 12.09 ng/ml) and superficial gastritis (26.96 +/- 20.17 ng/ml). But no significant difference was found between the CEA levels of gastric cancer and chronic atrophic gastritis (48.66 +/- 31.87 ng/ml). Also, elevated CEA was closely correlated to intestinal metaplasia. The positive rate of Ig was significantly higher in gastric cancer (IgG greater than or equal to 185 ug/ml, IgA greater than or equal to 100 ug/ml) than in benign gastric diseases. Although no correlation is present in the CEA and Ig in gastric juice, the combination of these two methods could improve the diagnostic accuracy. We believe that the two assays are worthy for screening gastric cancer from patients with high risk, and for identifying precancerous lesions.

Adult↗