Implication of the first and third extracellular loops of the mu-opioid receptor in the formation of the ligand binding site: a study using chimeric mu-opioid/angiotensin receptors.

Recent studies on chimeric mu/delta-, mu/kappa- and delta/kappa-opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric mu receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric mu receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100-fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than mu receptors, was shown to be responsible for this situation. Substitution of the mu receptor second extracellular loop by that of the angiotensin receptor diminished by approximately 10-fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the mu receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.

Agonist-induced signaling and trafficking of the mu-opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C-terminal domain.

The human mu-opioid receptor and a mutant form, muS/ T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C-terminal domain were changed to Ala, were studied after expression in CHO-K1 cells. Although the mutant receptors had similar affinities for agonists and EC50 values for inhibition of adenylyl cyclase as compared to wild-type receptors, the Emax were almost 2-fold decreased, suggesting a role of the mutated residues in G-protein coupling. After chronic morphine or etorphine, the EC50 values of the agonists were about 5-fold increased at both receptors but the Emax values were not altered; upon agonist withdrawal forskolin-stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down-regulation of the mu-opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the muS/T[i3+Cter]A receptor was not sequestered and was up-regulated (150-380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C-terminus of the mu-opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.

Identification in the mu-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents.

Inactivation by thiol reducing and alkylating agents of ligand binding to the human mu-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 mu 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 microM) 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 mu-opioid receptor.

Ionic and energetic changes at competence in the naturally transformable bacterium Streptococcus pneumoniae.

Addition of competence factor extracts to trigger competence in a culture of Streptococcus pneumoniae induced an increase in the intracellular pH and the Na+ content of the bacteria without any change in the K+ pool or in the membrane potential. These ionic shifts were concomitant with a stimulation of glycolysis that resulted in an enhanced ATP pool. Thus, in transforming conditions, at extracellular pH 7.8, competent bacteria presented a particularly high energetic state resulting from an increase in delta pH and in the ATP pool, associated with an enhanced Na+ content. These features are discussed in the context of homeostasis regulation in response to an environmental stimulus.