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Cloning, expression and classification of a kappa3-related opioid receptor using antisense oligodeoxynucleotides
217 CLONING, EXPRESSION AND CLASSIFICATION OF A KAPPA3-RELATED OPIOID RECEPTOR USING ANTISENSE OLIGODEOXYNUCLEOTIDES Pan, YX, Cheng, J, Xu, J, Pastemak, GW, The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, Departments &Neurology & Neuroscience and Pharmacology, CorneU U. Medical College, New York, NY 10021
Abstract: Using degenerate oligodeoxynucleotide primers based upon the predicted amino acid sequence of the cloned delta receptor in RT-PCR we isolated a cDNA of approximately 500 bases with high homology to the cloned delta, mu and kappa1 receptors. Before proceeding to clone the full length coding region, we designed an antisense oligodeoxynudeotide (ODN) and examined its actions in vivo. Administered intracerebroventricularly, the antisense ODN effectively blocks the analgesic actions of the kappaa agent naloxone benzoylhydrazone (NalBzoH) without affecting morphine (mu) analgesia. A mismatch ODN identical in base composition to the antisense but with four bases out of sequence is inactive. Having established the relevance of the sequence, we proceeded to clone the full length cDNA from a mouse brain library and characterize the expressed protein.
Prior work from our laboratory (1-4) and others (5) has suggested the existence of a kappa3 receptor with a unique binding and pharmacological profile. This receptor, with a density in the brain twice that of either mu or delta receptors, has a binding profile distinct from any previously described site and elicits analgesia through a novel mechanism. Kappa3 analgesia is readily reversed by general opioid antagonists such as WIN44,441, but it is insensitive towards antagonists selective for mu (naloxonazine and 13-FNA), delta (naltrindole) and kappa1 (norBNI) receptors. It also shows no cross tolerance with morphine (mu) or U50,488H (kappa1) analgesics. Using molecular biological methods based upon the recently cloned delta receptor (6,7), we now have identified a novel kappa3-related opioid receptor (8). Using degenerate oligodeoxynucleotide primers based upon the predicted amino acid sequence of the cloned delta receptor, we used a PCR approach to isolated a unique eDNA of approximately 500 bases with high homology to the cloned delta, mu and kappa1 receptors. We wished to establish the relevance &this sequence before attempting to clone the full length cDNA. Previously our group established the utility of antisense Kappa~ OliflodeoxynucleoUde Treatment oligodeoxyndeotides (ODN) in defining the :O% pharmacology of cloned delta (9), mu (10) and kappa 1 *p < O.D02 (1 l) receptors. This approach has been replicated (12). We designed an antisense oligodeoxynudeotide (ODN) of 20 bases and administered it (5 i~g, i.c.v.) on Days 1, 3 and 5 as previously described (9-11). On Day 6 we injected NalBzoH ( ~tg, i.c.v.) and examine analgesia in ~ i I..,,n. the tailflick assay 30 rain later (Fig. 1). As shown in the o% figure, the antisense ODN effectively blocks NalBzoH analgesia without affecting mu (morphine) analgesia. Additional studies (not shown) reveal that the same 0~, " , Morphine NalBzoH antisense is inactive against delta (DPDPE, i.t.) and kappa 1 (U50,488H, i.t.) analgesia as well. To further Figure 1: Blockadeofkappa, analgesiaby antisense. establish the specificity of the response, we designed a Analgesiais definedquantallyin the tailflickassayas a mismatch ODN which is identical in base composition to doublingor greaterof baseline latenciesand statistical significancewas determinedusingthe FisherExactTest, as the antisense but with four bases out of sequence. The previouslydescribed(8-10).
218 mismatch ODN is inactive. Having established the relevance of the sequence, we proceeded to clone the full length eDNA fi'om a mouse brain library and characterize the expressed protein. The clone codes for a protein of 367 amino acids and has high homology to delta, mu and kappa1 receptors at the amino aeide level and is similar to that recently reported (13). Doolittle-Kyte modeling predicts a seven membrane spanning receptor belonging to the G-protein receptor family. The expressed receptor in COS-7 cells is recognized by a monoclonal antibody raised against native kappa3 receptors in Western blots as a diffuse band of approximately 73 kdaltons. In vitro translation gives a single band which also is recognized by the antibody. Incubation of the in vitro translation product with microsomal membranes significantly increases the molecular weight, consistent with glycosylation. Although some opiates inhibit forskolin-stimulated eyclase in COS-7 cells expressing the receptor, binding has been difficult to demonstrate. In conclusion, we have identified a novel receptor highly homologous to previously established mu, delta and kappa~ receptors. Antisense studies implicate the protein in kappa3 analgesia and it is recognized by a selective monodonal antibody directed against native kappa3 receptors. However, the unusual pharmacological profile in cyclase studies and the difficulty in demonstrating binding preclude a definitive assignment as a kappa3 receptor.
Supported,in part, by grantsfrom N'IDAto GWP (DA02615 and DA00138). YXP is supportedby a Fellowshipfrom the Aaron Diamond Fundation. These results were presented,in part, at the N/DA TechnicalReview: "The MolecularNeurobiologyand PharmacologyofOpiateReceptorSubtypes:A tributeto WillimnMartin",heldin Washington,DC, November,1993 (7). The GenBank accessionnumber is U0942I. REFERENCES
1. M. Price, M.A.Gistrak, Y. Itzhak, E.F.Hahn and G.W. Pasternak (1989) Mol. Pharmacol. 35, 67-74. 2. J.A. Clark, L. Liu, M.Price, B.Hersh, M. Edelson and G.W. Pasternak J. Pharmacol. Exp. Ther. 251, 461-468, 1989. 3. M.A. Gistrak, D.Paul, E.F. Hahn and G.W. Pastemak (1989) J. Pharmacol. Exp. Ther. 251,469-476. 4. D. Paul, J.A. Levison, D.H. Howard, C.G. Pick, E.F. Hahn and G.W.Pasternak (1990) J. Pharmacol. Exp. Ther. 255, 769-774. 5. R.B. Rothman, V. Bykov, B,R. DeCosta, A.E, Jacobson, K.C. Rice, and L.S. Brady (1990) Peptides 11,311-317. 6. C. Evans, D.E. Keith Jr., H. Morrison, K. Magendzo and R.H. Edwards (1992) Science 258, 19521955. 7. B.L. Kieffer, K. Befort, C. Gaveriaux-Ruffand C.G. Hirth (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12048-12052. 8. G.R. Uhl, S.R. Childers and G.W. Pasternak (1994) Trends in Neursci. 17, 89-93. 9. K.M. Standifer, C.-C. Chien, C. Wahlestedt, G.P. Brown and G.W. Pasternak (1994) Neuron 12, 805810. 10. G. Rossi, Y.X. Pan, J. Cheng and G.W. Pasternak (1994) Life Sci. 54, PL375-379. 11. C.C. Chien, G.P. Brown, Y.X. Pan and G.W. Pasternak (1994) Eur. J. Pharmacol. 253, R7-8. 12. E.F. Bilsky, N. Bemstein, G.W. Pasternak, V.J. Hruby, D. Patel, F. Porreca and J. Lai, Life Sci. PL, in press. 13. C. Mollereau, M. Parmentier, P. Maiileux, J.L. Butour, C. Moisand, P. Chalon, D. Caput, G. Vassart and J.C. Meunier (1994) FEBS Letters 341, 33-38.
Abstract: Using degenerate oligodeoxynucleotide primers based upon the predicted amino acid sequence of the cloned delta receptor in RT-PCR we isolated a cDNA of approximately 500 bases with high homology to the cloned delta, mu and kappa1 receptors. Before proceeding to clone the full length coding region, we designed an antisense oligodeoxynudeotide (ODN) and examined its actions in vivo. Administered intracerebroventricularly, the antisense ODN effectively blocks the analgesic actions of the kappaa agent naloxone benzoylhydrazone (NalBzoH) without affecting morphine (mu) analgesia. A mismatch ODN identical in base composition to the antisense but with four bases out of sequence is inactive. Having established the relevance of the sequence, we proceeded to clone the full length cDNA from a mouse brain library and characterize the expressed protein.
Prior work from our laboratory (1-4) and others (5) has suggested the existence of a kappa3 receptor with a unique binding and pharmacological profile. This receptor, with a density in the brain twice that of either mu or delta receptors, has a binding profile distinct from any previously described site and elicits analgesia through a novel mechanism. Kappa3 analgesia is readily reversed by general opioid antagonists such as WIN44,441, but it is insensitive towards antagonists selective for mu (naloxonazine and 13-FNA), delta (naltrindole) and kappa1 (norBNI) receptors. It also shows no cross tolerance with morphine (mu) or U50,488H (kappa1) analgesics. Using molecular biological methods based upon the recently cloned delta receptor (6,7), we now have identified a novel kappa3-related opioid receptor (8). Using degenerate oligodeoxynucleotide primers based upon the predicted amino acid sequence of the cloned delta receptor, we used a PCR approach to isolated a unique eDNA of approximately 500 bases with high homology to the cloned delta, mu and kappa1 receptors. We wished to establish the relevance &this sequence before attempting to clone the full length cDNA. Previously our group established the utility of antisense Kappa~ OliflodeoxynucleoUde Treatment oligodeoxyndeotides (ODN) in defining the :O% pharmacology of cloned delta (9), mu (10) and kappa 1 *p < O.D02 (1 l) receptors. This approach has been replicated (12). We designed an antisense oligodeoxynudeotide (ODN) of 20 bases and administered it (5 i~g, i.c.v.) on Days 1, 3 and 5 as previously described (9-11). On Day 6 we injected NalBzoH ( ~tg, i.c.v.) and examine analgesia in ~ i I..,,n. the tailflick assay 30 rain later (Fig. 1). As shown in the o% figure, the antisense ODN effectively blocks NalBzoH analgesia without affecting mu (morphine) analgesia. Additional studies (not shown) reveal that the same 0~, " , Morphine NalBzoH antisense is inactive against delta (DPDPE, i.t.) and kappa 1 (U50,488H, i.t.) analgesia as well. To further Figure 1: Blockadeofkappa, analgesiaby antisense. establish the specificity of the response, we designed a Analgesiais definedquantallyin the tailflickassayas a mismatch ODN which is identical in base composition to doublingor greaterof baseline latenciesand statistical significancewas determinedusingthe FisherExactTest, as the antisense but with four bases out of sequence. The previouslydescribed(8-10).
218 mismatch ODN is inactive. Having established the relevance of the sequence, we proceeded to clone the full length eDNA fi'om a mouse brain library and characterize the expressed protein. The clone codes for a protein of 367 amino acids and has high homology to delta, mu and kappa1 receptors at the amino aeide level and is similar to that recently reported (13). Doolittle-Kyte modeling predicts a seven membrane spanning receptor belonging to the G-protein receptor family. The expressed receptor in COS-7 cells is recognized by a monoclonal antibody raised against native kappa3 receptors in Western blots as a diffuse band of approximately 73 kdaltons. In vitro translation gives a single band which also is recognized by the antibody. Incubation of the in vitro translation product with microsomal membranes significantly increases the molecular weight, consistent with glycosylation. Although some opiates inhibit forskolin-stimulated eyclase in COS-7 cells expressing the receptor, binding has been difficult to demonstrate. In conclusion, we have identified a novel receptor highly homologous to previously established mu, delta and kappa~ receptors. Antisense studies implicate the protein in kappa3 analgesia and it is recognized by a selective monodonal antibody directed against native kappa3 receptors. However, the unusual pharmacological profile in cyclase studies and the difficulty in demonstrating binding preclude a definitive assignment as a kappa3 receptor.
Supported,in part, by grantsfrom N'IDAto GWP (DA02615 and DA00138). YXP is supportedby a Fellowshipfrom the Aaron Diamond Fundation. These results were presented,in part, at the N/DA TechnicalReview: "The MolecularNeurobiologyand PharmacologyofOpiateReceptorSubtypes:A tributeto WillimnMartin",heldin Washington,DC, November,1993 (7). The GenBank accessionnumber is U0942I. REFERENCES
1. M. Price, M.A.Gistrak, Y. Itzhak, E.F.Hahn and G.W. Pasternak (1989) Mol. Pharmacol. 35, 67-74. 2. J.A. Clark, L. Liu, M.Price, B.Hersh, M. Edelson and G.W. Pasternak J. Pharmacol. Exp. Ther. 251, 461-468, 1989. 3. M.A. Gistrak, D.Paul, E.F. Hahn and G.W. Pastemak (1989) J. Pharmacol. Exp. Ther. 251,469-476. 4. D. Paul, J.A. Levison, D.H. Howard, C.G. Pick, E.F. Hahn and G.W.Pasternak (1990) J. Pharmacol. Exp. Ther. 255, 769-774. 5. R.B. Rothman, V. Bykov, B,R. DeCosta, A.E, Jacobson, K.C. Rice, and L.S. Brady (1990) Peptides 11,311-317. 6. C. Evans, D.E. Keith Jr., H. Morrison, K. Magendzo and R.H. Edwards (1992) Science 258, 19521955. 7. B.L. Kieffer, K. Befort, C. Gaveriaux-Ruffand C.G. Hirth (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12048-12052. 8. G.R. Uhl, S.R. Childers and G.W. Pasternak (1994) Trends in Neursci. 17, 89-93. 9. K.M. Standifer, C.-C. Chien, C. Wahlestedt, G.P. Brown and G.W. Pasternak (1994) Neuron 12, 805810. 10. G. Rossi, Y.X. Pan, J. Cheng and G.W. Pasternak (1994) Life Sci. 54, PL375-379. 11. C.C. Chien, G.P. Brown, Y.X. Pan and G.W. Pasternak (1994) Eur. J. Pharmacol. 253, R7-8. 12. E.F. Bilsky, N. Bemstein, G.W. Pasternak, V.J. Hruby, D. Patel, F. Porreca and J. Lai, Life Sci. PL, in press. 13. C. Mollereau, M. Parmentier, P. Maiileux, J.L. Butour, C. Moisand, P. Chalon, D. Caput, G. Vassart and J.C. Meunier (1994) FEBS Letters 341, 33-38.