The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of and ␦ receptors with G protein or ␤-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent G␤ 1 . In this system, the agonistinduced enhancement of BRET (indicating shortening of distance between the two proteins) was G␣-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at ␦ and receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (␦) or partial agonists () for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.Physiological agonists are usually equally efficient in promoting the interaction of receptors with G protein and arrestin, but manmade analogues can show divergent molecular efficacies for the two transducers (1, 2). This phenomenon, often addressed with a pictorial terminology (3-5), has attracted great interest and if better understood might lead to new types of drugs.The differential efficacy of opioids for G protein and arrestin interactions is also important in the mechanism of opiate addiction. As reported earlier, the addictive opiate morphine cannot induce and actually blocks desensitization and G protein uncoupling of ␦-opioid receptors (DOPR) 2 in neuroblastoma or in transfected cells (6, 7). Subsequent work shows that morphine is a poor inducer of rapid arrestin-dependent endocytosis for both ␦ and (MOPR) receptors (8 -11), although one exception is in striatum neurons with high levels of G protein receptor kinases (12).Two theories predict a relation between lack of endocytosis and the addiction liability of opioids, but the proposed explanations are radically different. One sees rapid endocytosis as a means to quench receptor signaling. Thus, the abnormally sustained signaling pattern produced by a drug that cannot internalize the receptor would promote post-receptor compensatory mechanisms, which may be responsible for the...

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

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We engineered single and multiple mutations of serines 203, 204, and 207 in the fifth transmembrane domain of the ␤ 2 -adrenergic receptor, a region known to interact with hydroxyl groups of the catechol ring. Using such mutants, we measured the binding affinities of a panel of six catecholamine agonists differing only in the presence of substituents in the ethanolamine tail of the molecule. Although all ligands shared an intact catechol ring, they exhibited different losses of binding energy in response to the mutations. For all mutations, we found a clear relationship between the loss of binding caused by receptor mutation and that caused by the ligand modification. This indicates that the catechol ring and the ethanolamine tail synergistically influence their respective interactions when binding to the receptor. To verify this idea by a formal thermodynamic test, we used a double-mutant cycle analysis. We compared the effects of each receptor mutation with those induced by the modifications of the ligand's tail. Because such changes disrupt interactions occurring at different receptor domains, they should produce cumulative losses. In contrast, we found positive cooperativity between such effects. This means that the binding of each side of the catecholamine can enhance the binding of the other, through an effect that is probably propagated via a conformational change. We suggest that the agonist-binding pocket is not rigid but is dynamically formed as the ligand builds an increasing number of contacts with the receptor."Induced fit" occurs during the binding of a protein to other proteins or small ligands. The term alludes to the notion that any binding process in proteins has a conformational consequence. In fact, intermolecular and intramolecular interactions in proteins depend on identical forces (Weber, 1972); thus, any change in one type of interactions must necessarily affect the other. Although the role of a flexible structure in ligand binding can be an essential part of protein function, its contribution to binding affinity is often neglected in the interpretation of site-directed mutagenesis studies. In this article, we evaluate the presence of induced-fit mechanisms in agonist binding to the ␤ 2 -adrenergic receptor, a typical member of the G protein-coupled receptor family, and exploit a strategy based on double mutant thermodynamic analysis to deduce to what extent agonist affinity is affected by conformation.Site-directed mutagenesis experiments in ␤ 2 -AR have identified important sites that may be forming the ligandbinding pocket for catecholamines. Two such sites, Asp 113 in TM3 and a cluster of serines (203, 204, and 207) in TM5 (Strader et al., 1988, 1989;Sato et al., 1999;Ambrosio et al., 2000;Liapakis et al., 2000), are located on separated domains of the molecule and hold, respectively, the amine tail, and the phenolic head of adrenergics. In a previous study (Del Carmine et al., 2002), we assessed how the deletion of both S204 and S207 in TM5 affected the binding on large n...

1 We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human b 2 adrenergic receptor (b 2 AR). 2 A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding a nities of more than 40 adrenergic ligands. Moreover, the e cacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type b 2 AR. 3 Although the two mutations had opposite e ects on ligand a nity, the extents of change were in both cases largely correlated with the degree of ligand e cacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the con®guration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4 We also found that AA mutation-induced a nity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of a nity that such modi®cations of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the bethanolamine tail with other residues of the b 2 -AR binding pocket.