Cloning and functional characterization through antisense mapping of a kappa 3-related opioid receptor.
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.
Pan
Y X
YX
Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Cheng
J
J
Xu
J
J
Rossi
G
G
Jacobson
E
E
Ryan-Moro
J
J
Brooks
A I
AI
Dean
G E
GE
Standifer
K M
KM
Pasternak
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000094942:Nociceptin Receptor
D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D000094942:Nociceptin Receptor; C000722287:Oprl1 protein, mouse