Purification of a Yellow Fluorescent Protein‐Tagged Mu Opioid Receptor and Functional Incorporation into rHDL Particles

Abstract

Despite extensive characterization of the opioid receptor much is unknown about the biochemical properties of the isolated receptor. Here we present the purification of the mu‐opioid receptor and demonstrate functional reconstitution into phospholipid bilayers. We constructed a human mu‐opioid receptor fused to yellow fluorescent protein at its amino‐terminus (YMOR). Baculovirus‐mediated expression of YMOR in insect cells yields receptors with high affinity [3H]diprenorphine (DPN) binding (Kd=0.5 nM). Dodecylmaltoside‐solubilized (with cholesterol hemisuccinate) YMOR was purified using a series of chromatography steps. Purified YMOR in detergent micelles showed impaired binding to [3H]DPN (Kd=4.14 nM), consistent with the behavior of many GPCRs in detergent. Incorporation of purified YMOR into a phospholipid bilayer in the form of reconstituted high density lipoprotein (rHDL) particles was able to restore high affinity [3H]DPN binding (0.26 nM). YMOR in rHDL bound naltrexone, naloxone and beta‐funaltrexamine with high affinity (Ki = 1.64, 11.9 and 2.8 nM, respectively). DAMGO‐stimulated [35S]GTPgammaS binding to Gi or Go is observed when YMOR is reconstituted into HDL. These data illustrate that the mu‐opioid receptor can be purified to homogeneity and its functional properties restored when incorporated into rHDL particles. This work was supported by NIGMS (GM068603).