Cloning and functional characterization through antisense mapping of a k3-related opioid receptor. Mol Pharmacol 47: 1180-1188
Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Molecular Pharmacology
(Impact Factor: 4.13).
We have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned mu, delta, and kappa 1 receptors. Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to mu, delta, and kappa 1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation in COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native kappa 3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region. Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of kappa 3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The mu, delta, and kappa 1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block kappa 3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. In conclusion, the antibody and antisense studies strongly associate KOR-3 with the kappa 3-opioid receptor, although it is not clear whether it is the kappa 3 receptor itself or a splice variant.
Available from: Courtney L Donica
- "The multiplicity of opioid receptor subtypes enables opiates and endogenous opioid peptides to elicit diverse physiologic and pharmacological actions. Before the cloning of the NOP receptor [also known as opioid receptor–like 1, KOR3, OP4 (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994; Pan et al., 1995)], the opioid receptor superfamily consisted of the classical MOR, DOR, and KOR. The NOP receptor sequence is approximately 50–60% identical to the classic opioid receptors ; however, neither endogenous opioid peptides nor selective MOR, KOR, and DOR ligands [with the exception of KOR agonist, dynorphin A (Zhang et al., 1998)] bind to or activate the NOP receptor (Bunzow et al., 1994; Chen et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994; Fukuda et al., 1997). "
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ABSTRACT: The Nociceptin/Orphanin FQ (N/OFQ) peptide (NOP) receptor is the fourth and most recently discovered member of the opioid receptor superfamily that also includes μ, δ and κ opioid receptor subtypes (MOR, DOR and KOR, respectively). The widespread anatomical distribution of the NOP receptor enables the modulation of several physiological processes by its endogenous agonist, N/OFQ. Accordingly, the NOP receptor has gained a lot of attention as a potential target for the development of ligands with therapeutic utility in several pathophysiological states. NOP receptor activation frequently results in effects opposing classical opioid receptor action, therefore regulation of the NOP receptor and conditions affecting its modulatory tone are important to understand. Mounting evidence illustrates a heterologous interaction of the NOP receptor with other G protein-coupled receptors, including MOR, DOR and KOR, which may subsequently influence their function. Our focus in this review is to summarize and discuss the findings that delineate the cellular mechanisms of NOP receptor signaling and regulation as well as the regulation of other receptors by N/OFQ and the NOP receptor.
Molecular pharmacology 02/2013; 83(5). DOI:10.1124/mol.112.084632 · 4.13 Impact Factor
Available from: Courtney L Donica
- "The NOP receptor was first cloned and identified as LC132 (Bunzow et al., 1994), opioid receptor-like 1 receptor (ORL-1; Mollereau et al., 1994), XOR1 (Chen et al., 1994), ROR-C (Fukuda et al., 1994) and KOR-3 (Pan et al., 1995). Its endogenous ligand, N/OFQ was identified within the next year by two groups. "
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ABSTRACT: Nociceptin/Orphanin FQ (N/OFQ) appears to contribute to the development of morphine tolerance, as blockade of its actions will block or reverse the process. To better understand the contribution of N/OFQ to the development of morphine tolerance, this study examined the effect of chronic morphine treatment on levels of N/OFQ and levels and activity of the N/OFQ peptide (NOP) receptor in spinal cord (SC) from male and female rats. Both male and female Wistar rats showed less responsiveness to morphine after subcutaneous injection of escalating doses of morphine (10, 20, 40, 60 and 80 mg/kg, respectively) twice daily for five consecutive days. Male rats were more tolerant to the antinociceptive actions of morphine than females. The N/OFQ content of SC extracts was higher in females than in males, regardless of treatment; following chronic morphine treatment the difference in N/OFQ levels between males and females was more pronounced. N/OFQ content in cerebrospinal fluid (CSF) was reduced 40% in male and 16% in female rats with chronic morphine exposure, but increased in periaqueductal grey of both sexes. Chronic morphine treatment increased NOP receptor levels 173% in males and 137% in females, while decreasing affinity in both. Chronic morphine increased the efficacy of N/OFQ-stimulated [³⁵S]GTPγS binding to SC membranes from male rats, consistent with increased receptor levels. Taken together, these findings demonstrate sex differences in N/OFQ-NOP receptor expression and NOP receptor activity following chronic morphine treatment. They also suggest interplay between endogenous N/OFQ and chronic morphine treatment that results in nociceptive modulation.
Neuropharmacology 05/2012; 63(3):427-33. DOI:10.1016/j.neuropharm.2012.04.028 · 5.11 Impact Factor
Available from: Grace Rossi
- "Northern blot analysis was performed as described [22,55]. Briefly, 20 μg of total brain RNA/lane was separated on a 0.8% formaldehyde agarose gel, and transferred to GenePlus membrane. "
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ABSTRACT: The mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat.
The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding.
The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various μ opioids seen in rats.
Molecular Pain 01/2011; 7(1):9. DOI:10.1186/1744-8069-7-9 · 3.65 Impact Factor
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