Purification and Functional Reconstitution of Monomeric ��-Opioid Receptors: ALLOSTERIC MODULATION OF AGONIST BINDING BY Gi2*

Despite extensive characterization of the μ-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A μ-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [³⁵S]guanosine 5′-(γ-thio)triphosphate binding to Gα_i, and is allosterically regulated by coupled G_i protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys⁷, Cys⁸]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the μ-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.Opioid receptors are members of the G protein-coupled receptor (GPCR)² superfamily and are clinical mainstays for inducing analgesia. Three isoforms of opioid receptors, μ, δ, and κ, have been cloned and are known to couple to G_i/o proteins to regulate adenylyl cyclase and K⁺/Ca⁺ ion channels (1–3). An ever growing amount of data suggests that many GPCRs oligomerize (4, 5), and several studies have suggested that μ-opioid receptors (MORs) and δ-opioid receptors heterodimerize to form unique ligand binding and G protein-activating units (6–10). Although intriguing, these studies utilize cellular overexpression systems where it is difficult to know the exact nature of protein complexes formed between the receptors.To study the function of isolated GPCRs, our laboratory and others have utilized a novel phospholipid bilayer reconstitution method (11–16). In this approach purified GPCRs are reconstituted into the phospholipid bilayer of a high density lipoprotein (HDL) particle. The reconstituted HDL (rHDL) particles are monodispersed, uniform in size, and preferentially incorporate a GPCR monomer (14, 15). Previous work in our lab has shown that rhodopsin, a class A GPCR previously proposed to function as a dimer (17–19), is fully capable of activating its G protein when reconstituted as a monomer in the rHDL lipid bilayer (15). Moreover, we have demonstrated that agonist binding to a monomeric β₂-adrenergic receptor, another class A GPCR, can be allosterically regulated by G proteins (14). This led us to determine whether a monomer of MOR, a class A GPCR that endogenously binds peptide ligands, is the minimal functional unit required to activate coupled G proteins. We additionally investigated whether agonist binding to monomeric MOR is allosterically regulated by inhibitory G protein heterotrimer.To study the function of monomeric MOR we have purified a modified version of the receptor to near homogeneity. A yellow fluorescent protein was fused to the N terminus of MOR, and this construct (YMOR) was expressed in insect cells for purification. After reconstitution of purified YMOR into rHDL particles, single-molecule imaging of Cy3-labeled and Cy5-labeled YMOR determined that the rHDL particles contained one receptor. This monomeric YMOR sample binds ligands with affinities nearly equivalent to those observed in plasma membrane preparations. Monomeric YMOR efficiently stimulates GTPγS binding to G_i2 heterotrimeric G protein. G_i2 allosteric regulation of agonist binding to rHDL·YMOR was also observed. Single-particle imaging of binding of [Lys⁷, Cys⁸]dermorphin-Cy3, a fluorophore-labeled agonist, to rHDL·YMOR supports the notion that the rHDL particles contain a single YMOR. Taken together, these results suggest that a monomeric MOR is the minimal functional unit for ligand binding and G protein activation and illustrate a novel method for imaging ligand binding to opioid receptors.     

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly

MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.     

Meeting report: SMART timing—principles of single molecule techniques course at the University of Michigan 2014

Four days after the announcement of the 2014 Nobel Prize in Chemistry for “the development of super‐resolved fluorescence microscopy” based on single molecule detection, the Single Molecule Analysis in Real‐Time (SMART) Center at the University of Michigan hosted a “Principles of Single Molecule Techniques 2014” course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 296–302, 2015.