|Home | About | Journals | Submit | Contact Us | Français|
Glycosylation of the μ-opioid receptor may play an important role on its function. Using nested PCR, N53Q mutation was prepared in the N-terminal region of the rat μ-opioid receptor cDNA and cloned into the pcDNA3 vector. The plasmids containing the wild-type and mutated receptor cDNA were transfected into the COS-7 cells. Intracellular cAMP was measured in the morphine-treated and untreated transfected cells using an ELISA kit. Plasmid expression was evaluated using X-gal staining. Intracellular concentration of cAMP in the N53Q-mutated cells was not significantly different from the wild-type. The expression of the transfected plasmids was confirmed. Therefore, based on these results, it seems that glycosylation at the N53 site of the rat μ-opioid receptor does not influence the function of this receptor significantly.
Opioid receptors belong to the superfamily of G-protein-coupled receptors (GPCRs), which consists of over 1000 members.1 They have seven transmembrane domains and one extracellular amino-terminal (N-terminal) with multiple glycosylation sites.2
Post-translational modifications such as glycosylation and palmitoylation frequently occur in these receptors, affecting their structure and function.1 Investigating these modifications can lead to a better understanding of the receptor properties; it also can be a guide to the design of better drugs and understanding inter-individual variations in drug response and may propose new strategies for prevention of tolerance and dependence.3–7
N-glycosylation is a common post-translational protein modification,8–10 which occurs on the amide group of an asparagine that is part of the consensus sequence of N-X-S/T (N, asparagine; S, serine; T, threonine; X, any amino acid except proline), known as the glycosylation consensus.8,11–16 Glycosylation has been shown to influence GPCRs differently, ranging from a decrease to an increase in the function of these proteins.3,13 Regarding opioid receptors, to our knowledge, only two site-directed mutagenesis studies have been performed in their amino terminal glycosylation sites.3,13 In both of these studies, asparagines (N) at position 40 was mutated to aspartate (D), i.e., N40D substitution, performed in human μ-opioid receptor cDNA.13 In one of these studies,13 the substitution led to an increase in affinity of β-endorphin and also potency of the ligand in activating potassium channels. The study also established that the N40D mutation was the most frequent one amongst heroin addicts.3 In the second study,3 it was reported that the N40D mutation did not cause any changes in affinity or potency of several μ-opioid receptor agonists or signal transduction. N40 is one of the five potential sites of glycosylation in the human μ-opioid receptor.3
Position 40 in the rat μ-opioid receptor (RMOR) is not a glycosylation site; thus, in the present study, the mutant receptor cDNA at the position of 53 (N53Q) in RMOR was constructed because of the proximity to N40 in the human μ-opioid receptor gene. Asparagine 53 of the RMOR was mutated to glutamine to evaluate the role of this glycosylation on the function of the receptor. We preferred to alter asparagine to glutamine (N to Q substitution), as these two amino acids are structurally very similar, contrary to asparagine and aspartic acid residues (N to D substitution used in the previous studies).8,12,15–17 Studying this mutation can not only help to clarify the differences observed in previous studies but can also shed more light on the role of glycosylation on μ receptor function.
PCR buffer (10×) and DNA Taq polymerase enzyme were from Bio-tools (Spain). dNTP was from Eurobio (France), and DNA ligase enzyme, glycerin, MOPS, agarose, alkaline phosphatase, and all of the restriction enzymes were from Roche (Germany). Chloroquine diphosphate, DEAE-dextran, forskolin, HBSS, 3-isobutyl-1-methylxanthine (IBMX), and penicillin–streptomycin were from Sigma-Aldrich (St. Louis, MO, USA). Ampicillin, DMEM, FBS, and trypsin were obtained from Gibco (Scotland). Bromophenol blue was from Fluka (St. Louis, MO, USA). λ DNA marker was purchased from Fermentas (Poland). Trypton and yeast extract were from Liofilchem (Italy). Ethidium bromide, boric acid, and loading dye buffer were from Cinnagen (Iran). Other materials were purchased from Merck (Germany) in molecular grade.
cAMP enzyme immunoassay (EIA) kit was from Assay Designs (Ann Arbor, MI, USA, USA), gel extraction kit from Qiagen (Germany), Midi plasmid preparation kit from Bio-Rad (Hercules, CA, USA), and TansformAid transformation kit from Fermentas.
The full-length cDNA clone encoding the RMOR (GenBank Accession Number L13069), subcloned in the pcDNA3 vector, was kindly provided by Professor Graeme Henderson (School of Medical Sciences, University of Bristol, Bristol, UK). The N53Q mutant (AAC to CAA substitution in the 157–158 position) was constructed using nested PCR as described previously.18
COS-7 cells (Pasteur Institute, I.R. Iran) were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell cultures were maintained at 37°C in a humidified 5% CO2 incubator.
To investigate μ-opioid receptor-mediated inhibition of forskolin-stimulated cAMP formation, COS-7 cells expressing μ-opioid receptors were seeded onto 24-well plates. On the experiment day, culture medium was removed, and cells were washed with serum-free medium. Then, cell cultures were incubated for 20 min at 37°C with 1 mM IBMX and 100 μM forskolin in the presence or absence of 10 μM morphine sulfate, a μ-opioid receptor agonist. The reaction was terminated by removing the media, the cells were then detached and lysed using 0.25% trypsin and 0.1 M HCl, and the cAMP level was determined using the cAMP EIA kit (Assay Designs). The cAMP kit uses a competitive immunoassay for the quantitative determination of the intracellular cAMP in the samples. The kit uses a polyclonal antibody to cAMP to bind competitively to the cAMP in the standard, sample, or the cAMP attached covalently to an alkaline phosphatase molecule. All of the standards and samples were run in duplicate. The OD of the samples and standards were read at λ = 405 nm. The intensity of the bound yellow color was inversely proportional to the concentration of the cAMP in standards or samples. The measured OD was used to calculate the concentration of cAMP.
The average net OD bound was calculated for each standard and sample by subtracting the average nonspecific binding (NSB) OD from average OD bound: average net OD = average bound OD − average NSB OD.
The curve of average net OD versus concentration of cAMP for the standards was obtained, a straight line was approximated through the points, and then, the concentration of cAMP in the unknown samples was determined by interpolation. Microsoft Excel software was used to analyze the dose-response curve for μ-opioid receptor-mediated inhibition of adenylyl cyclase activity.
All results are expressed as mean ± sem. The independent sample Student's t-test (two-tailed) was used to determine whether the difference was statistically significant (P = 0.05).
The mutation was produced using nested PCR, and the mutated cDNA was inserted into the pcDNA3 plasmid. Digestion of this construct resulted in the appearance of the expected insert size of about 500 bp (Fig. 1).
After transfection of COS-7 cells with the wild-type or mutated plasmids, X-gal staining was performed to detect expression of the receptor in the transfected cells. Nearly all of the transfected cells showed blue color, indicating that transfection was successful (Fig. 2A and B). No visible difference in cell staining between the wild-type and N53Q mutant-transfected cells was observed. These results are in agreement with what has been reported by other investigators.21–23
COS-7 cells were transiently transfected with the wild-type or N53Q mutant RMOR plasmids. Forty-eight hours after transfection, the cells were treated with IBMX (1 mM for 20 min) and then forskolin (100 μM) plus morphine sulfate (10 μM) for another 20 min. cAMP concentration in the wild-type group was greater than the N53Q mutant-transfected group (0.062 compared with 0.035 pmol/ml, respectively), but this difference was not statistically significant (Fig. 3).
The aim of the present study was to evaluate the role of glycosylation on the function of the RMOR. Transfection was successful according to the X-gal-staining method. Intracellular cAMP concentration was measured using a commercial kit and reported as pmol/ml, similar to other studies.24,25 The cAMP concentration in mutant group was not significantly different from the cells transfected with the wild-type receptor. This means that glycosylation at the N53 position may have no obvious effect on the function of the RMOR. On the contrary, Bond et al.13 showed that N40D substitution in human μ-opioid receptor increased the affinity of β-endorphin and also potency of the ligand in activating potassium channels as a target of the receptor activation. They used the Xenopus oocyte expression system. In the study,13 β-endorphin, a large endogenous opioid peptide, was used, and in the present study, morphine, a smaller alkaloid agonist, was used. Another difference is that potassium channel activity was used as their functional assay,13 and in the present study, reduction in cAMP production was measured. Additionally, cell lines used in the two studies were different; thus, altogether, these differences may contribute to the observed results.
Contrary to the results of Bond et al.13 and in support of our findings, Befort and colleagues3 concluded that the N40D mutant had similar affinity, potency, signaling, and down-regulation when compared with the wild-type receptor. They used COS-7 cells for the expression and functional assays as our study.3 In that study, several opioids (morphine, diprenorphine, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-amide) and opioid peptides (β-endorphin, [Met]enkephalin, and dynorphin A) were used and displayed similar binding affinities for the N40D mutant.3,13
Our study has an advantage over the investigations mentioned above, as N has been substituted to Q instead of D. Asparagine and glutamine are structurally more similar as compared with asparagine and aspartic acid.6 Thus, it is expected that only a distinct glycosylation site of the RMOR be interrupted, and the rest of the receptor characteristics would be preserved. Therefore, from what has been discussed, it can be concluded that N53 glycosylation of μ-receptor does not seem to influence its function significantly.
Regarding other GPCRs, it was shown that two out of the three N-glycosylation sites of the melanin-concentrating hormone receptor 1 were not necessary for its activity.6 This finding was similar to another study, in which the removal of three glycosylation sites of the gastrin-releasing peptide receptor did not alter receptor affinity and G-protein coupling.12
In contrast, Zhong et al. showed that the nonglycosylated P2Y12 receptor is defective in the inhibition of adenylyl cyclase activity.26 In another study, it was concluded that N-glycosylation of N4 or N16 was required for the expression of the functionally active tocopherol α isoform of the thromboxane A2 receptor.27 Other studies about the corticotropin-releasing factor receptor type 1, prostacyclin receptor, human thyrotropin receptor, human type 1 α metabotropic glutamate receptor, and somatostatin receptor have also shown that N-glycosylation has an important role in their function.8,10,16,28,29
In conclusion, in μ-opioid receptors, the glycosylation of the N53 position does not seem to influence the receptor function. However, based on the available data regarding other receptors and as the μ receptor has five glycosylation sites at its N terminal, the possibility of the importance of these sites on its expression and function cannot be ruled out. As some of the studies mentioned above indicate that a combination of mutations is needed to show the effects of glycosylation, it seems appropriate that in future studies, this strategy be applied to advance our understanding of this post-translational modification on μ-receptor function.
This work was supported by Isfahan University of Medical Sciences. The authors thank F. Moazzen and S. Sadeghi for their technical assistance.