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1.
We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca(1/2)) of the Ca-ATPase, 290 +/- 10 nM, is shifted to 580 +/- 20 nM when co-reconstituted with PLB(C) (Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLB(C) association with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa(1/2) upon reconstitution of the Ca-ATPase with P21A-PLB(C), which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLB(C) is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the alpha-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLB(C) are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLB(C) that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch. 相似文献
2.
Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB. 相似文献
3.
Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by beta-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 degrees C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 degrees C, has no inhibitory activity when assayed at 25 degrees C. However, at 37 degrees C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 degrees C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase. 相似文献
4.
Ren XQ Furukawa T Chen ZS Okumura H Aoki S Sumizawa T Tani A Komatsu M Mei XD Akiyama S 《Biochemical and biophysical research communications》2000,270(2):608-615
YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1. 相似文献
5.
Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions to modulate rate-limiting conformational transitions involving the transport activity of the Ca-ATPase. To investigate structural changes within the Ca-ATPase that result from the phosphorylation of PLB by cAMP-dependent protein kinase (PKA), we have covalently bound the long-lived phosphorescent probe erythrosin isothiocyanate (Er-ITC) to cytoplasmic sequences within the Ca-ATPase. Under these labeling conditions, the Ca-ATPase remains catalytically active, indicating that observed changes in rotational dynamics reflect normal conformational transitions. Two major Er-ITC labeling sites were identified using electrospray ionization mass spectrometry (ESI-MS), corresponding to Lys464 and Lys650, which are respectively located within the phosphorylation and nucleotide binding domains of the Ca-ATPase. Frequency-domain phosphorescence measurements of the rotational dynamics of Er-ITC bound to these cytoplasmic sequences within the Ca-ATPase permit the resolution of the dynamic structure of individual domain elements relative to the overall rotational motion of the entire Ca-ATPase polypeptide chain. We observe a significant decrease in the rotational dynamics of Er-ITC bound to the Ca-ATPase upon phosphorylation of PLB by PKA, as evidenced by an increase in the residual anisotropy. These results suggest that phosphorylation of PLB results in a structural reorientation of the phosphorylation or nucleotide binding domains with respect to the membrane normal. In contrast, calcium activation of the Ca-ATPase in the presence of dephosphorylated PLB results in no detectable change in the rotational dynamics of Er-ITC, suggesting that calcium binding and PLB phosphorylation have distinct effects on the conformation of the Ca-ATPase. We suggest that PLB functions to alter the efficiency of phosphoenyzme formation following calcium activation of the Ca-ATPase by modulating the spatial arrangement between ATP bound in the nucleotide binding domain and Asp351 in the phosphorylation domain. 相似文献
6.
Purification and characterization of aminopeptidase N from Spodoptera litura expressed in Sf21 insect cells 总被引:1,自引:0,他引:1
Insecticidal crystal proteins produced by strains of Bacillus thuringiensis cause larval death upon interaction with specific receptors located at the midgut epithelium of susceptible insects. Large quantities of easily purified aminopeptidase and cadherin-like Cry toxin receptors can facilitate the further study of Cry toxin binding and pore formation. Here, we report the solubilisation and purification of aminopeptidase N from Spodoptera litura (SlAPN). Recombinantly expressed and membrane anchored aminopeptidase N showed differential solubilisation with various ionic and nonionic detergents. The N-lauryl sarcosine (NLS)-solubilised SlAPN was purified to near homogeneity by anion exchange and gel filtration chromatography and refolded to its catalytically active form. The optimized purification regimen lead to >90% purification of the catalytically active SlAPN with 11% recovery and 9-folds purification. The interaction of purified SlAPN with biologically active Cry1C protein has been qualitatively and quantitatively characterized. By ligand blotting experiment, we demonstrated the linearity of interaction of the two purified proteins and lack of interaction of SlAPN with structurally divergent nontoxic Cry1Ac protein. The equilibrium dissociation constant (K(D)) of purified SlAPN for Cry1C was calculated by ELISA (90nM). Interaction of enzymatically inactive SlAPN with Cry1C and catalytic activity of APN-Cry1C complex suggested that the catalytic site and toxin-binding sites of SlAPN do not overlap. 相似文献
7.
Bruce A. Davis Istvan Edes Ramesh C. Gupta Ellen F. Young Hae Won Kim Nancy A. E. Steenaart Grazyna Szymanska Evangelia G. Kranias 《Molecular and cellular biochemistry》1990,99(2):83-88
The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca2+-calmodulin-dependent, and 3) Ca2+-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is also associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium. 相似文献
8.
Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni(2+) affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed. 相似文献
9.
A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads. 相似文献
10.
Kurisu M Morita M Kashiwayama Y Yokota S Hayashi H Sakai Y Ohkuma S Nishimura M Imanaka T 《Biochemical and biophysical research communications》2003,306(1):169-176
Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids. 相似文献
11.
Said M Vittone L Mundina-Weilenmann C Ferrero P Kranias EG Mattiazzi A 《American journal of physiology. Heart and circulatory physiology》2003,285(3):H1198-H1205
Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 microM) or the beta-adrenergic blocker dl-propranolol (1 microM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role. 相似文献
12.
Pneumonia caused by Pneumocystis jirovecii is still a major opportunistic infection among patients with AIDS. This opportunitistic pathogen is susceptible to therapy with inhibitors of the enzyme dihydrofolate reductase (DHFR) that target cell growth. Recent studies have shown that recombinant human-derived Pneumocystis DHFR (pDHFR) differs from rat-derived pDHFR by 38% in amino acid sequence. However, characterization of drug susceptibility, kinetics, and the three-dimensional structure of human-derived pDHFR has been hampered by the limited availability of purified material. The present study was undertaken to develop procedures to prepare sufficient enzyme for structure/function studies. Protein yield was limited when human-derived pDHFR was expressed in Escherichia coli using a pET28a(+) vector with an N-terminal His-tag for the 25 kDa protein. Therefore, the protein was expressed in Sf21 insect cells by baculovirus infection. The soluble enzyme was purified from cell lysates via Ni-chelated chromatographic columns, yielding about 5.1 mg of human-derived pDHFR fusion protein per liter of Sf21 culture. The purified protein had the expected mass as determined from Western blots with antibody for the N-terminal His-tag. This His-tagged recombinant DHFR from human-derived Pneumocystis was catalytically active and demonstrated kinetics similar to the recombinant enzyme from rat-derived Pneumocystis. The present studies for production of soluble human-derived pDHFR indicated that the baculovirus expression system supported production of significantly purer catalytically active enzyme in higher yields than that expressed in bacterial cultures. These protocols now make it possible to facilitate screening of antifolates with selectivity for human-derived pDHFR and to determine its three-dimensional structure. 相似文献
13.
Dendritic cells (DCs) act as sentinels in peripheral tissues, continuously scavenging for antigens in their immediate surroundings. Their involvement in T cell responses is generally thought to consist of a linear progression of events, starting with capture of antigen in peripheral tissues such as the skin followed by migration to draining lymphoid organs and MHC-restricted presentation of antigen-derived peptide to induce T cell priming. The role of tissue-derived DCs in the direct priming of immune responses has lately been challenged. It now appears that, at least in some instances, a non-migratory subtype of DCs in the secondary lymphoid tissue presents tissue-derived antigen to T cells. Here, we review recent developments in research on DC function in the priming of immune responses. 相似文献
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15.
《Plant Physiology and Biochemistry》2003,41(6-7):619-627
Plant serine acetyltransferase (SAT, E.C. 2.3.1.30) catalyses the first connecting reaction between nitrogen/carbon and sulphate metabolism. SAT is associated with the second committed enzyme, O-acetylserine(thiol)lyase (OASTL, E.C. 4.2.99.8), in a bi-enzyme complex called cysteine synthase (CS). Metabolic regulation of SAT-bound OASTL in the presence of cysteine (Cys) is analysed with the extracts from the leaf cell compartments of Pisum sativum. To this end, a high performance liquid chromatography (HPLC) technique is developed to measure the rate of O-acetylserine (OAS) formation by SAT. Under physiological experimental conditions, L-Cys specifically inhibits chloroplast-SAT activity, which is linked to the sulphate assimilation network. This metabolic feedback control does not apply to the SAT activity located in the cytosol. The non-physiological range of L-Cys inhibits the mitochondrial isoform. L-Cys in a non-competitive manner in presence of L-serine or acetyl-CoA (Ki of 12–20 μM) inhibits partially purified chloroplast SAT, free of bound OASTL. The Ki values are in the range of Cys concentrations estimated in this compartment. Furthermore, we report for the first time that the multi-enzyme complex, CS dissociates in the presence of Cys as previously described with OAS. From this study, and with the integration of data previously reported in the literature, we hypothesize a new model for the regulation of Cys synthesis in plant cells containing a chloroplastic Cys-sensitive SAT. 相似文献
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17.
A cDNA clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins (M(r) 74 kDa and 64 kDa) reactive with anti-cholesterol esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 micrograms/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of cholesterol esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 micrograms/ml at Day 8. Both the 74- and the 64-kDa cholesterol esterases were biologically active and hydrolyzed the artificial substrate p-nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic cholesterol esterase. The recombinant enzyme will be useful for further characterization of this protein. 相似文献
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19.
Egger M.; Ruknudin A.; Niggli E.; Lederer W. J.; Schulze D. H. 《American journal of physiology. Cell physiology》1999,276(5):C1184
The mechanism ofNi2+ block of theNa+/Ca2+exchanger was examined in Sf 9 cells expressing the human heartNa+/Ca2+exchanger (NCX1-NACA1). As predicted from the reported actions ofNi2+, its application reducedextracellular Na+-dependentchanges in intracellular Ca2+concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied bymeasured63Ni2+entry. The63Ni2+entry was observed in Sf 9 cells expressing theNa+/Ca2+exchanger but not in control cells. The established sequential transport mechanism of theNa+/Ca2+exchanger could be compatible with these results if one of the two iontranslocation steps is blocked byNi2+ and the other permitsNi2+ translocation. We concludethat, because Ni2+ entry wasinhibited by extracellular Ca2+and enhanced by extracellular Na+,the Ca2+ translocation step movedNi2+, whereas theNa+ translocation step wasinhibited by Ni2+. A model ispresented to discuss these findings. 相似文献
20.
Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists. 相似文献