Cloning and functional characterization through antisense mapping of a kappa 3-related opioid receptor.
ABSTRACT 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.
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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 · 4.12 Impact Factor
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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 · 4.82 Impact Factor
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ABSTRACT: Although alterations in micro-opioid receptor (microOR) signaling mediate excitatory effects of opiates in opioid tolerance, the molecular mechanism for the excitatory effect of acute low dose morphine, as it relates to microOR coupling, is presently unknown. A pronounced coupling of microOR to the alpha subunit of G inhibitory protein emerged in periaqueductal gray (PAG) from mice systemically administered with morphine at a dose producing acute thermal hyperalgesia. This coupling was abolished in presence of the selective microOR antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) administered at the PAG site, showing that the low dose morphine effect is triggered by microOR activated G inhibitory protein at supraspinal level. When Gbetagamma downstream signalling was blocked by intra-PAG co-administration of 2-(3,4,5-trihydroxy-6-oxoxanthen-9-yl)cyclohexane-1-carboxylic acid, a compound that inhibits Gbetagamma dimer-dependent signaling, a complete prevention of low dose morphine induced acute thermal hyperalgesia was obtained. Phospholipase C beta3, an enzyme necessary to morphine hyperalgesia, was revealed to be associated with Gbetagamma in PAG. Although opioid administration induces a shift in microOR-G protein coupling from Gi to Gs after chronic administration, our data support that this condition is not realized in acute treatment providing evidence that a separate molecular mechanism underlies morphine induced acute excitatory effect.Journal of Neurochemistry 08/2009; 111(1):171-80. DOI:10.1111/j.1471-4159.2009.06308.x · 4.24 Impact Factor