11 research outputs found
Identification in the μ-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents
AbstractInactivation by thiol reducing and alkylating agents of ligand binding to the human μ-opioid receptor was examined. Dithiothreitol reduced the number of [3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the μ receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N-ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC50=20 μM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC50=2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N-ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the μ-opioid receptor
Expression and pharmacological characterization of the human μ-opioid receptor in the methylotrophic yeast Pichia pastoris
AbstractThe human μ-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae α-mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase-1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell). Competition studies with non-selective, and μ-, δ- and κ-selective opioid agonists and antagonists revealed a typical μ-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes
Processing of the dynamin Msp1p in S. pombe reveals an evolutionary switch between its orthologs Mgm1p in S. cerevisiae and OPA1 in mammals
AbstractMitochondrial fusion depends on the evolutionary conserved dynamin, OPA1/Mgm1p/Msp1p, whose activity is controlled by proteolytic processing. Since processing diverges between Mgm1p (Saccharomyces cerevisiae) and OPA1 (mammals), we explored this process in another model, Msp1p in Schizosaccharomyces pombe. Generation of the short isoform of Msp1p neither results from the maturation of the long isoform nor correlates with mitochondrial ATP levels. Msp1p is processed by rhomboid and a protease of the matrix ATPase associated with various cellular activities (m-AAA) family. The former is involved in the generation of short Msp1p and the latter in the stability of long Msp1p. These results reveal that Msp1p processing may represent an evolutionary switch between Mgm1p and OPA1
The BH3‐only Bnip3 binds to the dynamin Opa1 to promote mitochondrial fragmentation and apoptosis by distinct mechanisms
Mitochondrial dynamics and disease, OPA1
AbstractThe mitochondria are dynamic organelles that constantly fuse and divide. An equilibrium between fusion and fission controls the morphology of the mitochondria, which appear as dots or elongated tubules depending the prevailing force. Characterization of the components of the fission and fusion machineries has progressed considerably, and the emerging question now is what role mitochondrial dynamics play in mitochondrial and cellular functions. Its importance has been highlighted by the discovery that two human diseases are caused by mutations in the two mitochondrial pro-fusion genes, MFN2 and OPA1. This review will focus on data concerning the function of OPA1, mutations in which cause optic atrophy, with respect to the underlying pathophysiological processes