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P Y Law

Publications and source records attributed to P Y Law.

At least 19 recordsLinked to original sources
PubMed2006

Disease-associated Glut1 single amino acid substitute mutations S66F, R126C, and T295M constitute Glut1-deficiency states in vitro.

Glucose transporter type 1 deficiency syndrome (Glut1DS) is the result of autosomal-dominant loss-of-function mutation of the glucose transporter type 1 gene (GLUT1) leading to brain energy failure and epileptic encephalopathy. In this study, the protein products of the Glut1DS-associated GLUT1 missense mutations, S66F, R126C, and T295M, were characterized using the Glut1-green fluorescent protein (GFP) fusion expressed in CHO cells. Glut1-GFP expression was confirmed by Western blot and confocal microscopy. The applicability of this Glut1-GFP expression model in reporting Glut1 functional deficits was validated by re-confirming the glucose transport defects of the previously reported pathogenic mutations R126H, R126L, and R333W. While S66F, R126C, and T295M mutants were expressed and targeted to the cell membrane, these Glut1 mutants have significantly diminished membrane association and glucose transport activity (p<0.05) relative to the wild-type Glut1 protein. Consistent with the reduced Glut1 membrane association, glucose transport kinetics studies showed that S66F, R126C, and T295M mutants have significantly reduced (p<0.05) Vmax but not Km. Thus, Glut1 single amino acid substitute mutants S66F, R126C, and T295M impair glucose transport function and constitute Glut1-deficiency states in vitro. These results support the pathogenicity of Glut1 S66F, R126C, and T295M in vivo.

Amino Acid Substitution↗
PubMed2006

Adenylyl cyclase superactivation induced by long-term treatment with opioid agonist is dependent on receptor localized within lipid rafts and is independent of receptor internalization.

Long-term opioid agonist treatment results in adenylyl cyclase superactivation. A recent "RAVE" theory implicates a direct correlation between the ability of agonist to induce receptor internalization and the magnitude of adenylyl cyclase superactivation. We decided to test such a theory by examining the adenylyl cyclase superactivation after long-term activation of mu-opioid receptor (MOR) in an EcR293 cell model. We examined the magnitudes of adenylyl cyclase superactivation in the presence of naloxone after long-term treatment with morphine, etorphine, and methadone, three agonists reported to have differential activities in promoting MOR internalization. It can be shown that the magnitudes of adenylyl cyclase superactivation after treating with these three agonists, although different, were dependent on MOR density. Blunting MOR internalization with the dominant-negative mutant of dynamin, K44E, did not alter the magnitude of either morphine- or etorphine-induced adenylyl cyclase superactivation. In the presence of diprenorphine, the magnitude of adenylyl cyclase superactivation after etorphine treatment was identical to that observed with morphine. It could be demonstrated further that adenylyl cyclase superactivation is dependent on the cell surface-located MOR. Sucrose gradient fractionation demonstrated the colocalization of MOR and adenylyl cyclase V/VI with caveolin-1, a marker for lipid rafts. After long-term agonist treatment, the majority of MOR remained at the lipid rafts. Methyl-beta-cyclodextrin (MbetaCD) completely blunted the adenylyl cyclase superactivation and agonist-induced receptor internalization. These MbetaCD actions were reversed by incubating the cells with cholesterol. Thus, the adenylyl cyclase superactivation is not dependent on agonist-induced receptor internalization. Rather, the location of MOR at lipid rafts is an absolute requirement for the observed adenylyl cyclase superactivation.

Adenylyl Cyclases↗
PubMed2004

Inhibition of akt/protein kinase B signaling by naltrindole in small cell lung cancer cells.

The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic delta opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3beta and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.

Apoptosis↗
PubMed2004

Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence.

Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the mu-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.

Animals↗
PubMed2003

Ligand-selective activation of mu-oid receptor: demonstrated with deletion and single amino acid mutations of third intracellular loop domain.

The mechanism for the differential regulation of the mu-opioid receptor by agonists is investigated by identifying the receptor domains used to define the relative efficacies of three mu-opioid receptor-selective agonists: [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and [N-MePhe3,D-Pro4]-morphiceptin (PL017) to inhibit forskolin-stimulated intracellular cAMP production in human embryonic kidney 293 cells. This was accomplished by systematically deleting four to five amino acids clusters within the third intracellular loop of rat mu-opioid receptor, Arg258 to Arg280, followed by Ala substitution and scanning studies of the 276RRITR280 sequence, the putative G protein-coupling motif. Deletion of the four to five amino acid clusters resulted in differential effects on the affinities of the agonists and antagonists, and also on the potencies and coupling efficiencies of the three opioid agonists. Ala scanning studies of the 276RRITR280 sequence revealed also the differences between [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and PL017. Substitution of Arg276 or Ile278 with Ala reduced the potency of DAMGO but not that of morphine PL017. Meanwhile, mutation of Thr279 to Ala increased the potencies of morphine and PL017 but not that of DAMGO. The I278A mutation decreased the DAMGO coupling efficiency but increased the PL017 coupling efficiency. The R280A mutation resulted in the increase in PL017 potency and coupling efficiency without altering those of DAMGO and morphine. Thus, these mutation studies suggested that the activation of mu-opioid receptor and interaction between the critical domains such as RRITR within third intracellular loop and the G proteins are agonist-selective.

Adenylyl Cyclases↗
PubMed2001

Phosphorylation of Ser363, Thr370, and Ser375 residues within the carboxyl tail differentially regulates mu-opioid receptor internalization.

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.

Alanine↗
PubMed2001

Affinity labels as tools for the identification of opioid receptor recognition sites.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

Affinity Labels↗
PubMed2000

Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization.

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.

Amino Acid Sequence↗
PubMed2000

Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor.

Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the delta-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the delta-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations </=90 fmol/mg of protein were rapid desensitizations (t(12) <10 min) observed. Apparently, deltorphin II-induced receptor desensitization involves cellular events in addition to receptor phosphorylation. Mutation of Ser(363) in the carboxyl tail of the delta-opioid receptor to Ala completely abolished the deltorphin II-induced receptor phosphorylation but not the desensitization response. Although the magnitude of desensitization was attenuated, the rate of deltorphin II-induced receptor desensitization remained the same in the S363A mutant as compared with wild type. Also, the S363A mutant could internalize in the presence of deltorphin II. Only when the agonist-induced clathrin-coated pit-mediated receptor internalization was blocked by 0.4 m sucrose that the deltorphin II-induced receptor desensitization was abolished in the S363A mutant. Similarly, 0.4 m sucrose could partially block the agonist-induced rapid desensitization in HEK293 cells expressing the wild type delta-opioid receptor. Taken together, these data supported the hypothesis that rapid desensitization of the delta-opioid receptor involves both the phosphorylation and the internalization of the receptor.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies.

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.

Animals↗
PubMed2000

Receptor density and recycling affect the rate of agonist-induced desensitization of mu-opioid receptor.

Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for mu-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the mu-opioid receptor level with beta-furnaltrexamine (betaFNA). The reduction of the receptor level with 1 microM betaFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with betaFNA. Hence, mu-opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the mu-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of mu-opioid receptor desensitization in HEK293 cells.

Brefeldin A↗
PubMed2000

Molecular mechanisms and regulation of opioid receptor signaling.

Cloning of multiple opioid receptors has presented opportunities to investigate the mechanisms of multiple opioid receptor signaling and the regulation of these signals. The subsequent identification of receptor gene structures has also provided opportunities to study the regulation of receptor gene expression and to manipulate the concentration of the gene products in vivo. Thus, in the current review, we examine recent advances in the delineation basis for the multiple opioid receptor signaling, and their regulation at multiple levels. We discuss the use of receptor knockout animals to investigate the function and the pharmacology of these multiple opioid receptors. The reasons and basis for the multiple opioid receptor are addressed.

Adenylyl Cyclases↗
PubMed2000

delta-Opioid receptors are more efficiently coupled to adenylyl cyclase than to L-type Ca(2+) channels in transfected rat pituitary cells.

Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2+) channels.

Adenylate Cyclase Toxin↗
PubMed1999

Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure.

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Antibody Specificity↗
PubMed1999

The absence of a direct correlation between the loss of [D-Ala2, MePhe4,Gly5-ol]Enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation.

Chronic activation of the mu-opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the receptor. It has been suggested that opioid receptor phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent phosphorylation of the receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the receptor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapid rate of receptor phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between receptor phosphorylation and the loss of response was further demonstrated when receptor phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR1TAG phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) receptors were expressed in the same system, overexpression of beta-adrenergic receptor kinase 2 elevated agonist-induced phosphorylation for both receptors. However, in the same cell lines under the same conditions, overexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced receptor desensitization. Thus, these data show that phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the receptor.

Adenylyl Cyclase Inhibitors↗