A pharmacological comparison of the cloned frog and human mu opioid receptors reveals differences in opioid affinity and function

Abstract

This study presents a direct comparison of the ligand binding and signaling profiles of a mammalian and non-mammalian mu opioid receptor. Opioid ligand binding and agonist potencies were determined for an amphibian (Rana pipiens) mu opioid receptor (rpMOR) and the human mu opioid receptor (hMOR) in transfected, intact Chinese hamster ovary (CHO) cells. Identical conditions were employed such that statistically meaningful differences between the two receptors could be determined. Identifying these differences is an important first step in understanding how evolutionary changes affect ligand binding and signaling in vertebrate opioid receptors. As expected, the rank of opioid ligand affinity for rpMOR and hMOR was consistent with the ligands' previously characterized type-selectivity. However, most of the opioid ligands tested had significant differences in affinity for rpMOR and hMOR. For example, the mu-selective agonist, DAMGO ([d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin), had a 10.9-fold greater affinity (K(i)) for hMOR (K(i)=268 nM) than rpMOR (K(i)=2914 nM). In addition, differences in signaling between these receptors were found by measuring inhibition of cAMP accumulation by morphine or DAMGO. DAMGO was significantly more potent (13.6-fold) in CHO cells expressing hMOR versus those expressing rpMOR. In addition, a significantly greater maximal inhibition was elicited by both opioid agonists in cells expressing hMOR. In summary, this study supports an ongoing effort to better understand how vertebrate evolution has shaped opioid receptor properties and function.

Fig. 1

[³H]-Naloxone saturation binding in intact, whole CHO cells transiently expressing rpMOR (A) or hMOR (B). In rpMOR-CHO, [³H]-naloxone bound to a single site with a K_d of 0.29 nM and a B_max of 354 fmol/mg of protein. In hMOR-CHO, [³H]-naloxone bound to a single site with a K_d of 1.0 nM and a B_max of 641 fmol/mg of protein. Specific binding for each concentration of [³H]-naloxone was determined in triplicate in 3−4 independent experiments (error bars indicate S.E.M.).

Fig. 2

[³H]-Naloxone displacement by multiple opioid agonists in intact, whole CHO cells transiently expressing rpMOR (A) or hMOR (B). Agonists displaced [³H]-naloxone and bound to a single site. The K_i and Hill slope for each is shown in Table 1. Percent specific binding of [³H]-naloxone at each concentration of competitor was determined in triplicate in 3−4 independent experiments (error bars indicate S.E.M.).

Fig. 3

[³H]-Naloxone displacement by multiple opioid antagonists in intact, whole CHO cells transiently expressing rpMOR (A) or hMOR (B). Antagonists displaced [³H]-naloxone and bound to a single site. The K_i and Hill slope for each is shown in Table 1. Percent specific binding of [³H]-naloxone at each concentration of competitor was determined in triplicate in 3−4 independent experiments (error bars indicate S.E.M.).

Fig. 4

Inhibition of [³H]cAMP accumulation by morphine (A) or DAMGO (B) in CHO cells stably expressing rpMOR or hMOR. A range of opioid agonist concentrations were used in order to determine the concentration for 50% inhibition (IC₅₀) and maximal inhibition (I_max). Individual values are reported in Table 2. [³H]-cAMP accumulation is plotted as percent versus that in cells treated with 10 μM forskolin only. For each condition, 3−5 independent experiments were performed in triplicate (error bars indicate S.E.M.).

Fig. 5

ClustalW alignment of rpMOR and hMOR amino acid sequences. Alignment was done using default values (Mega v. 4.0, www.megasoftware.net). Identical sites are noted by white text with black background, conservative substitutions are noted by grey background, and different sites are boxed on the rpMOR sequence. .