Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors

Abstract

Despite extensive characterization of the -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 -opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [35 S]guanosine 5-(␥-thio)triphosphate binding to G␣ 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, [Lys 7 , Cys 8 ]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 -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, , ␦, and , have been cloned and are known to couple to G i/o proteins to regulate adenylyl cyclase and K ϩ /Ca ϩ ion channels(1-3). An ever growing amount of data suggests that many GPCRs oligomerize (4, 5), and several studies have suggested that -opioid receptors (MORs) and ␦-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)(18)(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 ␤ 2 -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 ...

Light on the structure of thromboxane A2 receptor heterodimers

“…Nonetheless, the mutant TPs that reached the plasma membrane bound the antagonist [ 3 H]-SQ29,548 with normal affinity, and could be activated by the stable TXA 2 agonist U46619 as efficiently as the wild-type receptors at equal level of expression. These data are consistent with the observation that monomeric GPCRs are the minimal functional unit in G protein activation and that dimerization/oligomerization is not absolutely required for this process [9][10][11][12][13][14]43]. However, the U46619 agonist, stimulated TM1 mutant TPa and TPb with a marked reduction in potency, thus suggesting that dimer formation favors a more efficient signaling complex.…”

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

“…State-Purified GPCRs have been reported to exhibit the characteristic interaction between agonists and guanylyl nucleotides after reconstitution in two different states: as monomers in nanodiscs, in the case of the ␤ 2 -adrenergic and -opioid receptors (21,23), and as tetramers in liposomes, in the case of the M 2 muscarinic receptor (6, 7). Both preparations exhibit at least two classes of sites, as distinguished by agonists, and saturating concentrations of the nucleotide promote their apparent interconversion from higher to lower affinity.…”

“…Purified monomers of some GPCRs 6 have been reconstituted either as monomers in nanodiscs of modified apolipoprotein A1 (20,21) or as oligomers in liposomes (4,6,7,22). Both preparations exhibit the expected specificities for their respective ligands; inconveniently, however, both preparations also exhibit an allosteric interaction between agonists and guanylyl nucleotides that is recognized as a hallmark of efficacy (6,7,21,23).…”

“…Both the ␤ 2 -adrenergic receptor and the -opioid receptor have been reconstituted as monomers in nanodiscs (21,23). In the absence of G protein, sites labeled by a radiolabeled antagonist were of uniformly low affinity for agonists.…”

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

“…It is well known from in vitro and in vivo experiments that GPCR monomers can recognize/decode signals. In this respect, worth mentioning are studies in which the monomeric entities of three class A GPCRs (namely rhodopsin, β 2 -adrenergic, and μ-opioid receptors) trapped into nanodiscs were able to signal as monomers (Bayburt et al, 2007;Whorton et al, 2007;Kuszak et al, 2009). Furthermore, signaling from GPCR monomers is characterized by an intrinsic plasticity, as GPCR activation can result in different patterns of signal transduction, such as G protein and/or arrestin pathways (Zidar et al, 2009).…”