Convergent Balancing Selection on the Mu-Opioid Receptor in Primates

Abstract

The mu opioid receptor is involved in many natural processes including stress response, pleasure, and pain. Mutations in the gene also have been associated with opiate and alcohol addictions as well as with responsivity to medication targeting these disorders. Two common and mutually exclusive polymorphisms have been identified in humans, A118G (N40D), found commonly in non-African populations, and C17T (V6A), found almost exclusively in African populations. Although A118G has been studied extensively for associations and in functional assays, C17T is much less well understood. In addition to a parallel polymorphism previously identified in rhesus macaques (Macaca mulatta), C77G (P26R), resequencing in additional non-human primate species identifies further common variation: C140T (P47L) in cynomolgus macaques (Macaca fascicularis), G55C (D19H) in vervet monkeys (Chlorocebus aethiops sabeus), A111T (L37F) in marmosets (Callithrix jacchus), and C55T (P19S) in squirrel monkeys (Saimiri boliviensis peruviensis). Functional effects on downstream signaling are observed for each of these variants following treatment with the endogenous agonist β-endorphin and the exogenous agonists morphine, DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin), and fentanyl. In addition to demonstrating the importance of functional equivalency in reference to population variation for minority health, this also shows how common evolutionary pressures have produced similar phenotypes across species, suggesting a shared response to environmental needs and perhaps elucidating the mechanism by which these organism-environment interactions are mediated physiologically and molecularly. These studies set the stage for future investigations of shared functional polymorphisms across species as a new genetic tool for translational research.

Keywords: balancing selection; molecular evolution; opioid; primates.

Fig. 1.

Overview of OPRM1 structure. (A)OPRM1 protein structure bound to β-funaltrexamine after PDB ID:4DKL (Manglik et al. 2012). (B) Exonic structure of OPRM1. Note that there are numerous alternative splice forms most of which incorporate alternate fourth exons. (C) Schematization of transmembrane domains after UniProt (Hinz 2010). (D). Prediction of secondary structure disorder (Yang et al. 2005).

Fig. 2.

Unrooted cladogram showing divergence and polymorphism in primate OPRM1. Divergence is shown on branches with amino acid substitutions above the line and synonymous substitutions below the lines. Polymorphism is shown at the terminus of branches again with amino acid mutations on top and synonymous mutations below. Polymorphisms have MAFs >0.1%, those in parentheses have MAFs >5%. (A) Shows values for the N-terminal domain of OPRM1. (B) Shows values for the remainder of the protein.

Fig. 3.

Effects of OPRM1 polymorphisms on β-endorphin-induced inhibition of cAMP formation. HEK cells transduced with an inducible CRE-responsive firefly luciferase reporter and stably expressing the target mu-opioid receptors were treated with 1 µM forskolin and varying concentrations of β-endorphin. For human mu-opioid receptor, human β-endorphin was used while for rhesus macaque, marmoset, and squirrel monkey mu-opioid receptor the non-ape simian β-endorphin was uses. Concentration response functions showing effects of species-specific pairwise variation are shown. EC₅₀ and E_max values are shown with significance calculated using an extra sum of squares F-test.

Fig. 4.

Effects of OPRM1 polymorphisms on fentanyl-induced inhibition of cAMP formation. HEK cells transduced with an inducible CRE-responsive firefly luciferase reporter and stably expressing the target mu-opioid receptors were treated with 1 µM forskolin and varying concentrations of fentanyl. Concentration response functions showing effects of species-specific pairwise variation are shown. EC₅₀ and E_max values are shown with significance calculated using an extra sum of squares F-test.

Fig. 5.

Effects of OPRM1 polymorphisms on morphine-induced inhibition of cAMP formation. HEK cells transduced with an inducible CRE-responsive firefly luciferase reporter and stably expressing the target mu-opioid receptors were treated with 1 µM forskolin and varying concentrations of morphine. Concentration response functions showing effects of species-specific pairwise variation are shown. EC₅₀ and E_max values are shown with significance calculated using an extra sum of squares F-test.

Fig. 6.

Effects of OPRM1 polymorphisms on DAMGO-induced inhibition of cAMP formation. HEK cells transduced with an inducible CRE-responsive firefly luciferase reporter and stably expressing the target mu-opioid receptors were treated with 1 µM forskolin and varying concentrations of DAMGO. Concentration response functions showing effects of species-specific pairwise variation are shown. EC₅₀ and E_max values are shown with significance calculated using an extra sum of squares F-test.