Characterization of Irreversible Binding of β-Funaltrexamine to the Cloned Rat μ Opioid Receptor

Chen, Chongguang; Xue, Jie; Zhu, Jinmin; Chen, Yung-Wu; Kunapuli, Satya P.; Riel, J K de; Yu, Lei; Liu‐Chen, Lee‐Yuan

doi:10.1074/jbc.270.30.17866

Cited by 73 publications

(45 citation statements)

References 24 publications

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“…m-Opioid receptors in the rat brain were shown to have molecular masses ranging from 60 to 75 kDa, and indicative of a glycoprotein nature that, upon deglycosylation, down-shifts to a sharp band of 39 kDa (for review see Chen et al 1995). The existence of a glycosylated form of the m-opioid receptor was predicted based on previous reports in the literature (in Chinese hamster ovary (CHO) cells and in human embryonic kidney (HEK)-293 cells (Chaturvedi et al 2001)) and on the presence of a typical N-glycosylation consensus sequence located at the N terminus (Chaturvedi et al 2000).…”

Section: Discussionmentioning

confidence: 99%

Expression and subcellular localization of the μ-opioid receptor in equine spermatozoa: evidence for its functional role

Albrizio

Guaricci²,

Maritato³

et al. 2005

Reproduction

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The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the m-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds m agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nucleotide conservation. By immunofluorescence, m-opioid receptor labeling was found on the sperm head and on the tail and disappeared in the acrosomal region of acrosome-reacted sperm cells. Immunoblotting revealed two bands of 50 and 65 kDa. Effects of the opioid antagonist naloxone on motility and on viability and capacitation/acrosome reaction were investigated by computerassisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10 23 M naloxone (P < 0.05), whereas it increased significantly after 5 h in 10 28 M naloxone (P < 0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10 23 M naloxone (P < 0.05), but increased significantly in the presence of 10 28 M (P < 0.001). Curvilinear velocity and amplitude of lateral head displacement in spermatozoa incubated in the presence of naloxone were not indicative of hyperactivation. H258/CTC staining showed that 10 28 M naloxone significantly stimulated capacitation (P < 0.01) after 3 h. However, it had no effect on sperm cell viability and acrosomal status. Overall, this study provides the first evidence that the m-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.

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Section: Discussionmentioning

confidence: 99%

Expression and subcellular localization of the μ-opioid receptor in equine spermatozoa: evidence for its functional role

Albrizio

Guaricci²,

Maritato³

et al. 2005

Reproduction

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“…The mixture was centrifuged and the pellets were washed three times by centrifugation and resuspension. Immunoprecipitated materials were dissolved in 2ϫ Lammeli sample buffer and subjected to 8% SDS-polyacrylamide gel electrophoresis (Chen et al, 1995), and 32 P was detected by use of a phosphoimager (Cyclone; PerkinElmer Life Sciences). Quantitation of receptor phosphorylation was performed with the OptiQuant software program.…”

Section: Methodsmentioning

confidence: 99%

Differential Regulation of the Human κ Opioid Receptor by Agonists: Etorphine and Levorphanol Reduced Dynorphin A- and U50,488H-Induced Internalization and Phosphorylation

Zhang²,

Jin³

et al. 2003

J Pharmacol Exp Ther

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We previously observed that (trans)-3,4-dichloro-N-methyl-N- [2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488H) promoted internalization and phosphorylation of the FLAG-tagged human opioid receptor (FLAG-hkor) stably expressed in Chinese hamster ovary (CHO) cells. In this study, we compared regulation of the FLAG-hkor expressed in CHO cells by U50,488H, dynorphin A, etorphine, and levorphanol, which were potent full agonists as determined by stimulation of guanosine 5Ј-O-(3-[ 35 S]thio)triphosphate binding. Using fluorescence flow cytometry, we found that dynorphin A(1-17), like U50,488H, promoted internalization of the FLAG-hkor in a time-and dose-dependent manner. The antagonists naloxone and norbinaltorphimine, having no effect on FLAG-hkor internalization, effectively blocked dynorphin A(1-17)-and U50,488H-induced internalization. Interestingly, the full agonists etorphine and levorphanol did not cause internalization of the FLAG-hkor but significantly reduced dynorphin A(1-17)-and U50,488H-induced internalization in a dose-dependent manner. Immunofluorescence staining of FLAG-hkor yielded similar results. Dynorphin A(1-17) and U50,488H enhanced phosphorylation of FLAG-hkor to a greater extent than etorphine, but levorphanol did not increase FLAG-hkor phosphorylation. Etorphine or levorphanol decreased dynorphin-or U50,488H-induced phosphorylation. It is likely that conformations of the hkor required for phosphorylation and initiation of internalization are different from those for activation of G proteins. We also examined whether the four agonists had differential effects on superactivation of adenylate cyclase. Pretreatment with U50,488H, dynorphin A(1-17), or etorphine enhanced forskolin-stimulated adenylate cyclase activity to ϳ200 to 250% of the control, whereas levorphanol pretreatment did not result in significant adenylate cyclase superactivation. Thus, the degree of superactivation caused by an agonist is unrelated to its ability to promote internalization of the hkor.

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“…However, this amino acid might not be a binding pocket for the opioid alkaloids, since using the / receptor chimeras indicated that ␤FNA alkylated amino acids within the region of TM6, e3 loop, and TM7. 83 The irreversible alkylation of the Lys 233 within the opioid receptor apparently was caused by the specific orientation of the fumaramate side chain within the tertiary structure of the receptor.…”

mentioning

confidence: 99%

Mutational analysis of the structure and function of opioid receptors

1999

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Characterization of Irreversible Binding of β-Funaltrexamine to the Cloned Rat μ Opioid Receptor

Cited by 73 publications

References 24 publications

Expression and subcellular localization of the μ-opioid receptor in equine spermatozoa: evidence for its functional role

Expression and subcellular localization of the μ-opioid receptor in equine spermatozoa: evidence for its functional role

Differential Regulation of the Human κ Opioid Receptor by Agonists: Etorphine and Levorphanol Reduced Dynorphin A- and U50,488H-Induced Internalization and Phosphorylation

Mutational analysis of the structure and function of opioid receptors

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