Signaling complexes of voltage-gated calcium channels and G protein-coupled receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (1, 2). Unlike the three types of classical mu, delta, and kappa opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (3). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (4). Similar signaling complexes between N-type channels and GABA(B) receptors have been reported (5). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (6, 7). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.     

Signaling Complexes of Voltage-Gated Calcium Channels and G Protein-Coupled Receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (). Unlike the three types of classical μ, δ, and κ opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (). Similar signaling complexes between N-type channels and GABA_B receptors have been reported (). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.     

Multiple opiate receptors: emerging concepts

Increasing biochemical evidence indicates that the wide spectrum of opiate pharmacological actions are mediated via heterogeneous classes of receptors. μ receptors have been identified as the high affinity sites where morphine-like opiates exert their analgesic effects. δ receptors have a somewhat different CNS distribution and have been identified as sites relatively selective for the naturally occuring enkephalins. Recent biochemical studies provide evidence for two additional classes of opiate receptor sites which were originally proposed on the basis of physiological studies. Ketocyclazocine-like opiates produce their unique ataxic and sedative effects via interaction with K receptors, and SKF-10,047 (N-allylnorcyclazocine) and related opiates produce stimulant and psychotomimetic effects via interactions with σ receptors.Many opiate drugs interact at multiple receptor sites. Thus, the constellation of neuropharmacological actions of a particular opioid ligand may reflect its various potencies at a combination of μ, δ, K, and σ receptors.     

Opioid elevation of intracellular free calcium: possible mechanisms and physiological relevance

Opioid receptors are seven transmembrane domain Gi/G0 protein-coupled receptors, the activation of which stimulates a variety of intracellular signalling mechanisms including activation of inwardly rectifying potassium channels, and inhibition of both voltage-operated N-type Ca2+ channels and adenylyl cyclase activity. It is now apparent that like many other Gi/G0-coupled receptors, opioid receptor activation can significantly elevate intracellular free Ca2+ ([Ca2+]i), although the mechanism underlying this phenomenon is not well understood. In some cases opioid receptor activation alone appears to elevate [Ca2+]i, but in many cases it requires concomitant activation of Gq-coupled receptors, which themselves stimulate Ca2+ release from intracellular stores via the inositol phosphate pathway. Given the number of Ca2+-sensitive processes known to occur in cells, there are therefore a myriad of situations in which opioid receptor-mediated elevations of [Ca2+](i) may be important. Here, we review the literature documenting opioid receptor-mediated elevations of [Ca2+]i, discussing both the possible mechanisms underlying this phenomenon and its potential physiological relevance.     

Exploring the structure of opioid receptors with homology modeling based on single and multiple templates and subsequent docking: A comparative study

Opioid receptors are the principal targets for opioids, which have been used as analgesics for centuries. Opioid receptors belong to the rhodopsin family of G-protein coupled receptors (GPCRs). In the absence of crystal structures of opioid receptors, 3D homology models have been reported with bovine rhodopsin as a template, though the sequence homology is low. Recently, it has been reported that use of multiple templates results in a better model for a target having low sequence identity with a single template. With the objective of carrying out a comparative study on the structural quality of the 3D models based on single and multiple templates, the homology models for opioid receptors (mu, delta and kappa) were generated using bovine rhodopsin as single template and the recently deposited crystal structures of squid rhodopsin, turkey β-1 and human β-2 adrenoreceptors along with bovine rhodopsin as multiple templates. In this paper we report the results of comparison between the refined 3D models based on multiple sequence alignment (MSA) and models built with bovine rhodopsin as template, using validation programs PROCHECK, PROSA, Verify 3D, Molprobity and docking studies. The results indicate that homology models of mu and kappa with multiple templates are better than those built with only bovine rhodopsin as template, whereas, in many aspects, the homology model of delta opioid receptor with single template is better with respect to the model based on multiple templates. Three nonselective ligands were docked to both the models of mu, delta and kappa opioid receptors using GOLD 3.1. The results of docking complied well with the pharamacophore, reported for nonspecific opioid ligands. The comparison of docking results for models with multiple templates and those with single template have been discussed in detail. Three selective ligands for each receptor were also docked. As the crystallographic structures are not yet known, this comparison will help in choosing better homology models of opioid receptors for studying ligand receptor interactions to design new potent opioid antagonists.     

Receptor-receptor interactions as an integrative mechanism in nerve cells

Several lines of evidence indicate that interactions among transmission lines can take place at the level of the cell membrane via interactions among macromolecules, integral or associated to the cell membrane, involved in signal recognition and transduction. The present view will focus on this last subject, i.e., on the interactions between receptors for chemical signals at the level of the neuronal membrane (receptor-receptor interaction). By receptor-receptor interaction we mean that a neurotransmitter or modulator, by binding to its receptor, modifies the characteristics of the receptor for another transmitter or modulator. Four types of interactions among transmission lines may be considered, but mainly intramembrane receptor-receptor interactions have been dealt with in this article, exemplified by the heteroregulation of D2 receptors via neuropeptide receptors and A2 receptors. The role of receptor-receptor interactions in the integration of signals is discussed, especially in terms of filtration of incoming signals, of integration of coincident signals, and of neuronal plasticity.     

背根神经节神经元阿片受体和离子通道的研究进展

阿片及阿片受体与外周神经系统镇痛机制的研究,随着分子生物学技术的发展,已在受体的分子结构、形态学、分子药理学、离子通道和细胞内信号转导系统等方面取得了显著进展。μ、δ、κ阿片受体分子结构上的部分差异决定了它们各自的功能特征。三种受体在初级感觉神经元分布的比例不同,但都能介导细胞Ｃａ＾２＋通道的抑制和Ｋ＾＋电流增加及减少。阿片受体和通道之间由多种第二信使系统偶联。分子药理学研究表明它们还存在亚型受体     

Exploring the unique pharmacology of a novel opioid receptor, ZFOR1, using molecular modeling and the 'message-address' concept.

Previous studies have probed the structural basis of ligand selectivity in the mu, delta and kappa opioid receptors through the application of molecular modeling techniques in concert with the 'message-address' concept. Here, this approach was used in an attempt to rationalize the unique pharmacological profile of a recently cloned novel opioid receptor, ZFOR1 (ZebraFish Opioid Receptor 1). Specifically, a model of the transmembrane domains of ZFOR1 was constructed and used to explore the binding modes of various prototypical opioid ligands. The results show that the 'message' portion of the binding pocket of ZFOR1 is highly conserved; hence, the binding modes of non-selective opioid ligands are well preserved. In contrast, a small number of variant residues at the extracellular end of the binding pocket, particularly Lys288 (VI:26) and Trp304 (VII:03), are shown to create adverse steric interactions with all delta and kappa selective ligands examined, thereby disrupting their binding modes. These results are consistent with, and serve as an explanation for, the observed pharmacology of this receptor, lending support to both the validity of the 'message-address' concept itself and to the use of molecular modeling approaches in its application.     

Ligand-receptor and receptor-receptor interactions act in concert to activate signaling in the Drosophila toll pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Sp?tzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Sp?tzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Sp?tzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.     

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

Binding mode characterization of 6α- and 6β-N-heterocyclic substituted naltrexamine derivatives via docking in opioid receptor crystal structures and site-directed mutagenesis studies: Application of the ‘message–address’ concept in development of mu opioid receptor selective antagonists

Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, nonpeptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles. Both compounds displayed high binding affinity and selectivity to the mu opioid receptor. To further study the interaction of these two ligands with the three opioid receptors, the recently released opioid receptor crystal structures were employed in docking studies to further test our original hypothesis that the ligands recognize a unique ‘address’ domain in the mu opioid receptor involving Trp318 that facilitates their selectivity. These modeling results were supported by site-directed mutagenesis studies on the mu opioid receptor, where the mutants Y210A and W318A confirmed the role of the latter in binding. Such work not only enriched the ‘message–address’ concept, also facilitated our next generation ligand design and development.     

Phosphatidylinositol-3 kinase is distinctively required for mu-, but not kappa-opioid receptor-induced activation of c-Jun N-terminal kinase

Opioid receptors are the therapeutic targets of narcotic analgesics. All three types of opioid receptors (mu, delta and kappa) are prototypical G(i)-coupled receptors with common signaling characteristics in their regulation of intracellular events. Nevertheless, numerous signaling processes are differentially regulated by the three receptors. We have recently demonstrated that stimulation of delta-opioid receptor can up-regulate the activity of the c-Jun N-terminal kinase (JNK) in a pertussis toxin-sensitive manner (Kam et al. 2003; J. Neurochem. 84, 503-513). The present study revealed that the mu-opioid receptor could stimulate JNK in both SH-SY5Y cells and transfected COS-7 cells. The mechanism by which the mu-opioid receptor stimulated JNK was delineated with the use of specific inhibitors and dominant-negative mutants of signaling intermediates. Activation of JNK by the mu-opioid receptor was mediated through G beta gamma, Src kinase, son-of-sevenless (Sos), Rac and Cdc42. Interestingly, unlike the delta-opioid receptors, the mu-opioid receptor required phosphatidylinositol-3 kinase (PI3K) to activate JNK. The mu-opioid receptor-induced JNK activation was effectively inhibited by wortmannin or the coexpression of a dominant negative mutant of PI3K gamma. Like the delta-opioid receptor, activation of JNK by the kappa-opioid receptor occurred in a PI3K-independent manner. These studies revealed that the mu-opioid receptor utilize a distinct mechanism to regulate JNK.     

Ligand-induced,receptor-mediated dimerization and activation of EGF receptor

The EGF receptor mediates many cellular responses in normal biological processes and in pathological states. Recent structural studies reveal the molecular basis for ligand binding specificity and how ligand binding induces receptor dimerization. Receptor dimerization is mediated by receptor-receptor interactions in which a loop protruding from neighboring receptors mediates receptor dimerization and activation.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

Evidence for delta opioid receptor subtypes regulating adenylyl cyclase activity in rat brain

Opioid agonists selective for mu- or delta opioid receptors inhibit adenyl yl cyclase in membranes from rat caudate-putamen and nucleus accumbens. The presence of subtypes of delta opioid receptors has been suggested. In both brain regions we have found that the inhibition of adenylyl cyclase by DPDPE was more readily antagonized by 7-benzylidenenaltrexone (BNTX), than by naltriben. In contrast, the inhibitory effects of deltorphin-II and DSLET were more readily antagonized by naltriben, than by BNTX. neither naltriben nor BNTX significantly antagonized the effect of a mu selective agonist. These results suggest that inhibition of adenylyl cyclase in caudate-putamen and nucleus accumbens is regulated by two forms of delta-opioid receptor with ligand selectivities similar to those two forms proposed to mediate analgesic effect.     

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights into agonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.     

A simple mathematical model of cooperativity in receptor mosaics based on the "symmetry rule"

The phenomenon of receptor-receptor interactions was hypothesized about 20 years ago. It has been demonstrated by now that receptor-receptor interactions between G-protein coupled receptors (GPCRs) occur at plasma membrane level and result in the reciprocal modulation of their binding characteristics (i.e., cooperativity). One of the most important feature of this phenomenon is the concept of cluster of receptors, or receptor mosaic (RM). However, no proper mathematical approach has still been available to characterize RMs as far as their receptor composition, receptor topography and order of receptor activation inside the RM. This paper tries to fill the gap. A simple mathematical approach to the cooperativity in RMs formed by dimers of identical receptors and/or by iso-receptors is proposed. To this aim the so-called "symmetry rule" has been considered. This approach allows to describe by means of a simple energy function the effects of receptor composition (number of dimers), spatial organisation (respective location of the dimers) and order of activation (order according to which the single receptors are ligated) on the integrative cooperativity (index) of the RMs.     

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH₂ (C-36) and Dmt-c[D-Lys-Phe-p-CF₃-Phe-Asp]NH₂ (F-81). The ability of these peptides to cross the blood–brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.     

Spinal administration of selective opioid antagonists in amphibians: evidence for an opioid unireceptor

In mammals, opioids act by interactions with three distinct types of receptors: mu, delta, or kappa opioid receptors. Using a novel assay of antinociception in the Northern grass frog, Rana pipiens, previous work demonstrated that selective mu, delta, or kappa opioids produced a potent antinociception when administered by the spinal route. The relative potency of this effect was highly correlated to that found in mammals. Present studies employing selective opioid antagonists, beta-FNA, NTI, or nor-BNI demonstrated that, in general, these antagonists were not selective in the amphibian model. These data have implications for the functional evolution of opioid receptors in vertebrates and suggest that the tested mu, delta, and kappa opioids mediate antinociception via a single type of opioid receptor in amphibians, termed the unireceptor.     

Co-expressions of different opioid receptor types differentially modulate their signaling via G(16)

Combinations of two different types of opioid receptors - delta-, kappa-, mu-opioid receptors (DOR, KOR, and MOR) and opioid receptor-like receptor 1 (ORL(1)) - were co-expressed with the alpha subunit of G(16) in COS-7 cells, and the ability of various selective agonists to induce activation of phospholipase Cbeta was examined. Nociceptin/orphanin FQ-induced response was enhanced when ORL(1) was co-expressed with MOR or KOR but not DOR. The kappa-agonist U50,488H induced a modest inositol phosphate formation when KOR was expressed alone or with MOR, but the response was attenuated when co-expressing with either DOR or ORL(1). It is suggested that the co-expressions of two different opioid receptor types indeed modify their downstream signaling events.