The kappa-opioid receptor from human placenta: Hydrodynamic characteristics and evidence for its association with A G protein

Abstract

The κ nature of opioid binding sites in a brush border membrane (BBM) fraction from human placenta has been confirmed: these sites display considerably higher apparent affinity (K I = 1.2 nM) for the κ selective ligand U-50488 than they do for the μ and δ selective ligands [D-Ala 2, MePhe 4, Glyol 5] enkephalin (K I = 1.5–2 μM) and [D-Thr 2, Leu 5] enkephalyl-Thr (K I = 10–15 μM), respectively. The BBM fraction from human placenta was incubated either with the agonist 3H-etorphine or with the antagonist 3H-diprenorphine and subsequently solubilized with digitonin. The solubilized macromolecular radioactivity was found to behave as a homogenous entity both in molecular exclusion chromatography (app. r s = 6.1 nm) and in linear sucrose gradients (S 20.w = 12 S). Two lines of evidence indicated that the placental κ opioid receptor is capable of interacting with a guanine nucleotide regulatory (G) protein: (i) equilibrium binding of the agonist 3H-etorphine in the BBM fraction was clearly inhibited by 5′-guanylylimidodiphosphate (Gpp(NH)p), especially in the presence of Na + ions while binding of the antagonist 3H-diprenorphine was significantly less so and (ii) the sedimentation velocity of the κ opioid receptor was decreased down to about 10 S when the BBM fraction was prelabeled with radioligand in the presence of Gpp(NH)p prior to its solubilization with digitonin. The G protein that mediates the effect of Gpp(NH)p might be neither G s nor G i since no adenylate cyclase activity could be demonstrated in the BBM fraction from human placenta.