Characterization and Distribution of a Cloned Rat μ‐Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ‐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 (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β‐endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d‐Ala², N‐Me‐Phe⁴,Gly‐ol⁵]‐enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ⋙ U50,488 (K_i = 910 ± 0.78 nM) > [d‐Pen^2,5]‐enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ‐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 µM 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 μ‐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 μ‐opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ‐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.