Molecular cloning and expression of a rat kappa opioid receptor.
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ABSTRACT:
SUBMITTER: Li S
PROVIDER: S-EPMC1134604 | biostudies-other | 1993 Nov
REPOSITORIES: biostudies-other
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Project description:By screening a rat brain cDNA library using a cloned mu opioid receptor cDNA as probe, a clone was identified that is very similar to both the mu and delta opioid receptor sequences. Transient expression of this clone in COS-7 cells showed that it encodes a kappa opioid receptor, designated KOR-1, which is capable of high-affinity binding to kappa-selective ligands. Treatment of transfected cell membranes with bremazocine, a kappa-selective agonist, resulted in a 53% decrease in adenylate cyclase activity, indicating that this kappa opioid receptor displays inhibitory coupling to adenylate cyclase. Thus, one member from each of the three opioid receptor types, mu, kappa and delta, has been molecularly cloned. Analysis of sequence similarities among these opioid receptors, as well as between opioid receptors and other G-protein-coupled receptors, revealed regions of sequence conservation that may underlie the ligand-binding and functional specificities of opioid receptors.
| S-EPMC1134603 | biostudies-other
Project description:A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype.
| S-EPMC47691 | biostudies-other
Project description:BackgroundThe misuse of opioids stems, in part, from inadequate knowledge of molecular interactions between opioids and opioid receptors. It is still unclear why some opioids are far more addictive than others. The κ-opioid receptor (KOR) plays a critical role in modulating pain, addiction, and many other physiological and pathological processes. Butorphanol, an opioid analgesic, is a less addictive opioid with unique pharmacological profiles. In this study, we investigated the interaction between butorphanol and KOR to obtain insights into the safe usage of this medication.MethodsWe determined the binding affinity of butorphanol to KOR with a naltrexone competition study. Recombinant KORs expressed in mammalian cell membranes (Chem-1) were used for G-protein activation studies, and a human embryonic kidney-293 (HEK-293) cell line stably transfected with the human KOR was used for β-arrestin study as previously described in the literature. The effects of butorphanol on KOR internalization were investigated using mouse neuroblastoma Neuro2A cells stably transfected with mKOR-tdTomato fusion protein (N2A-mKOR-tdT) cells overexpressing KOR. The active-state KOR crystal structure was used for docking calculation of butorphanol to characterize the ligand binding site. Salvinorin A, a full KOR agonist, was used as a control for comparison.ResultsThe affinity of KOR for butorphanol is characterized by Kd of 0.1 ± 0.02 nM, about 20-fold higher compared with that of the µ-opioid receptor (MOR; 2.4 ± 1.2 nM). Our data indicate that butorphanol is more potent on KOR than on MOR. In addition, butorphanol acts as a partial agonist of KOR in the G-protein activation pathway and is a full agonist on the β-arrestin recruitment pathway, similar to that of salvinorin A. The activation of the β-arrestin pathway is further confirmed by KOR internalization. The in silico docking model indicates that both salvinorin A and butorphanol share the same binding cavity with the KOR full agonist MP1104. This cavity plays an important role in determining either agonist or antagonist effects of the ligand.ConclusionsIn conclusion, butorphanol is a partial KOR agonist in the G-protein activation pathway and a potent KOR full agonist in the β-arrestin recruitment pathway. The structure analysis offers insights into the molecular mechanism of KOR interaction and activation by butorphanol.
| S-EPMC7668422 | biostudies-literature
Project description:The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.
| S-EPMC10008561 | biostudies-literature
Project description:We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and approximately 300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5'-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig kappa-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of (125)I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the approximately 300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.
| S-EPMC117947 | biostudies-literature
Project description:In the present study, the effects and mechanism of action of U50,488H (a selective κ-opioid receptor agonist) on calcification of rat vascular smooth muscle cells (VSMCs) induced by β-glycerophosphate (β-GP) were investigated. VSMCs were isolated and cultured in traditional FBS-based media. A calcification model was established in VSMCs under hyperphosphatemia and intracellular calcium contents. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and lactate were detected in cell culture supernatants before and after treatment. Alizarin red staining was used to detect the degree of calcification of VSMCs. Expression levels of key molecules of osteogenic markers, fructose-2,6-biphosphatase 3 (PFKFB3), and proline hydroxylase 2 (PHD2), were determined using western blotting. Further, vascular calcification was induced by vitamin D3 plus nicotine in rats and isolated thoracic aortas, calcium concentration was assessed in rat aortic rings in vitro. We demonstrated that U50,488H inhibited VSMC calcification in a concentration-dependent manner. Moreover, U50,488H significantly inhibited osteogenic differentiation and ALP activity in VSMCs pretreated with β-GP. Further studies confirmed that PFKFB3 expression, LDH level, and lactate content significantly increased during calcification of VSMCs; U50,488H reversed these changes. PHD2 expression showed the opposite trend compared to PFKFB3 expression. nor-BNI or 3-PO abolished U50,488H protective effects. Besides, U50,488H inhibited VSMC calcification in rat aortic rings ex vivo. Collectively, our experiments show that κ-opioid receptor activation inhibits VSMC calcification by reducing PFKFB3 expression and lactate content, providing a potential drug target and strategy for the clinical treatment of vascular calcification.
| S-EPMC8202865 | biostudies-literature
Project description:BACKGROUND:The proteinase-activated receptor 4 (PAR4) is a G-protein-coupled receptor activated by proteases such as thrombin and trypsin. Although activation of PAR4 has been shown to modulate rat gastrointestinal motility, the rat PAR4 sequence was unknown until now. This study aimed to identify the rat PAR4 cDNA. RESULTS:The cDNA coding for the rat PAR4 homologue was cloned from the duodenum. Northern blots demonstrated a 3.0 kb transcript in the duodenum. Protein homology with mouse and human counterparts was 90% and 75% respectively. PAR4 is expressed predominantly in the esophagus, stomach, duodenum and the spleen. When expressed in COS cells, PAR4 is activated by trypsin (1 nM), thrombin (50 nM), mouse PAR4 specific peptide (500 microM) and a putative rat PAR4 specific activating peptide (100 microM), as measured by intracellular Ca2+-changes. CONCLUSIONS:We have identified and characterized cDNA encoding the rat PAR4 homologue. PAR4 is expressed predominantly in the upper gastrointestinal tract. It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.
| S-EPMC88883 | biostudies-literature
Project description:Modification of the furan ring of salvinorin A (1), the main active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. Conversion of the furan ring to an aldehyde at the C-12 position (5) has allowed for the synthesis of analogues with new carbon-carbon bonds at that position. Previous methods for forming these bonds, such as Grignard and Stille conditions, have met with limited success. We report a palladium catalyzed Liebeskind-Srogl cross-coupling reaction of a thioester and a boronic acid that occurs at neutral pH and ambient temperature to produce ketone analogs at C-12. To the best of our knowledge, this is the first reported usage of the Liebeskind-Srogl reaction to diversify a natural product scaffold. We also describe a one-step protocol for the conversion of 1 to 12-epi-1 (3) through microwave irradiation. Previously, this synthetically challenging process has required multiple steps. Additionally, we report in this study that alkene 9 and aromatic analogues 12, 19, 23, 25, and 26 were discovered to retain affinity and selectivity at kappa opioid receptors (KOP). Finally, we report that the furan-2-yl analog of 1 (31) has similar affinity to 1. Collectively, these findings suggest that different aromatic groups appended directly to the decalin core may be well tolerated by KOP receptors, and may generate further ligands with affinity and activity at KOP receptors.
| S-EPMC3344910 | biostudies-literature
Project description:Opioid agonists produce analgesia in humans and other mammals by binding to three distinct types of G protein-coupled receptors; mu (MOR), delta (DOR), and kappa (KOR) opioid receptors. A fourth member of the opioid receptor family is the nociceptin or orphanin FQ receptor (ORL), however the role of the ORL receptor in analgesia is less clear. In the Northern grass frog, Rana pipiens, systemic and central administration of morphine and selective MOR, DOR, and KOR agonists produced dose-dependent antinociceptive effects blocked by the general opioid antagonist, naltrexone. The present study reports on the sequence, expression, and bioinformatics of four opioid receptor cDNAs cloned from Rana pipiens; rpMOR, rpDOR, rpKOR, and rpORL. These were the first opioid receptors cloned from a species of Class Amphibia, are selectively expressed in brain tissue, and show 70-84% identity to their homologous mammalian opioid receptors. Comparisons within species showed that MOR, DOR, and KOR proteins are significantly less divergent in earlier-evolved vertebrates compared to humans and other mammals. Among the four types of opioid receptors, MOR proteins show the least sequence variation among the six vertebrate species. Additionally, phylogenetic analysis supports the hypothesis that the family of opioid receptor proteins are coded by four genes that arose from two gene duplications of a single ancestral opioid receptor gene.
| S-EPMC3075437 | biostudies-literature
Project description:The κ opioid receptor (κOR) is a member of G-protein-coupled receptors, and is considered as a promising drug target for treating neurological diseases. κOR selective 6'-GNTI was proved to be a G-protein biased agonist, whereas 5'-GNTI acts as an antagonist. To investigate the molecular mechanism of how these two ligands induce different behaviors of the receptor, we built two systems containing the 5'-GNTI-κOR complex and the 6'-GNTI-κOR complex, respectively, and performed molecular dynamics simulations of the two systems. We observe that transmembrane (TM) helix 6 of the κOR rotates about 4.6(o) on average in the κOR-6'-GNTI complex. Detailed analyses of the simulation results indicate that E297(6.58) and I294(6.55) play crucial roles in the rotation of TM6. In the simulation of the κOR-5'-GNTI system, it is revealed that 5'-GNTI can stabilize TM6 in the inactive state form. In addition, the kink of TM7 is stabilized by a hydrogen bond between S324(7.47) and the residue V69(1.42) on TM1.
| S-EPMC4705513 | biostudies-literature