Characterization and distribution of a cloned rat mu-opioid receptor.
We have cloned and expressed a rat brain cDNA, TS11, that encodes a mu-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 +/- 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 +/- 0.2 nM) approximately beta-endorphin (Ki = 0.7 +/- 0.05 nM) approximately morphine (Ki = 0.8 +/- 0.5 nM) approximately [D-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; Ki = 1.6 +/- 0.5 nM) uch much greater than U50,488 (Ki = 910 +/- 0.78 nM) > [D-Pen2,5]- enkephalin (Ki = 3,170 +/- 98 nM) > dextrorphan (Ki = 4,100 +/- 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a mu-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) in COS-7 cells 50 microM 5'-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 +/- 0.5 nM) to a lower-affinity state (IC50 = 89.0 +/- 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned mu-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the mu-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of mu-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.
Bunzow
J R
JR
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Zhang
G
G
Bouvier
C
C
Saez
C
C
Ronnekleiv
O K
OK
Kelly
M J
MJ
Grandy
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology; D005260:Female; D017403:In Situ Hybridization; D007981:Levorphanol / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000032:analysis / Q000235:genetics / Q000502:physiology; D017473:Receptors, Opioid, kappa / Q000032:analysis / Q000235:genetics / Q000502:physiology; D017450:Receptors, Opioid, mu / Q000032:analysis* / Q000235:genetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D004745:Enkephalins; D012333:RNA, Messenger; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D007981:Levorphanol; D000242:Cyclic AMP