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

Abstract

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, -, ␦-, and -opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The -opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of -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.ORL1 (opioid receptor-like 1) receptors (also known as NOP or nociceptin receptors) belong to the class of G␣ i/o -linked seven-helix transmembrane receptors (1, 2). They are structurally related to members of the classical opioid receptor family (i.e. -, ␦-, and -opioid receptors) but do not interact with known opioid receptor agonists or antagonists. Instead, they are activated by the endogenous ligand orphanin FQ (also known as nociceptin), a 17-amino acid polypeptide (3). ORL1 receptors are expressed in both the central and peripheral nervous systems; the physiological effects of receptor activation include stimulation of food intake, reduced anxiety, reduced withdrawal symptoms, and when activated peripherally, analgesia (4 -6). There is a functional cross-talk between ORL1 receptors and -opioid receptors such that chronic administration of the -receptor agonist morphine results in increased ORL1 receptor expression levels (7), whereas knock-out of the ORL1 receptor gene results in decreased morphine tolerance (8) without compensatory changes in opioid receptor expression (9). This may hint at the possibility of overlapping mechanisms controlling cellular expression levels of these two receptor subtypes.We have recently shown that ORL1 receptors physically interact with N-type calcium channels and that this interaction results in two distinct consequences: first, an agonist-independent inhibition of N-type channels due to constitutive receptor activity (10), and second, receptor-mediated trafficking of N-type channels to and from the plasma membrane (11). Neither of these phenomena appeared to occur with -opioid receptors (11). ORL1 receptors have also been shown to heterodimerize with -opioid receptors (12), with dimerized receptors showing altered sensitiv...

G Protein Regulation of Neuronal Calcium Channels: Back to the Future

“…Similarly, a Ca v 2.2-opioid receptor like-1 (ORL-1; also known as nociception receptor) signaling complex has been documented in small DRG neurons, and supports a tonic agonist-independent G protein inhibition of the Ca 21 channel evidenced by prepulse facilitation (Beedle et al, 2004). Similar observations have been reported for m-and d-opioid receptors transiently expressed with Ca v 2.2 channel in tsA201 cells, although the existence of these protein complexes remains to be explored in native conditions (Chee et al, 2008;Evans et al, 2010). Also, a physical interaction exists between Ca v 2.2 channels and dopamine D1 and D2 receptors and requires other channel structural determinants, including the II-III intracellular linker Kisilevsky and Zamponi, 2008;Weiss, 2009).…”

“…However, internalization of Ca v 2.2 channels is not accompanied by a diminution of the membrane Ca 21 current, questioning the physiologic relevance of this regulation (Murali et al, 2012). In addition, although m-opioid receptors also physically interact with Ca v 2.2 channels, they do not cointernalize, indicating that biochemical coupling of the channel with the GPCR is not sufficient to mediate agonist-mediated internalization of the Ca 21 channel (Evans et al, 2010). It is also possible that the assembly of Ca v 2 channels and GPCRs provides a mechanism that ensures spatiotemporal regulation of the Ca 21 entering synaptic nerve terminals.…”

“…It was proposed that association of the Ca 21 channels with GPCRs might control channel density at the plasma membrane, providing an additional level of control of the Ca 21 influx. Indeed, activation of ORL-1 receptors triggers an agonist-dependent cointernalization of Ca v 2.2-ORL-1 complexes into vesicular compartments both in tsA201 cells and DRG neurons (Altier et al, 2006;Evans et al, 2010). However, internalization of Ca v 2.2 channels is not accompanied by a diminution of the membrane Ca 21 current, questioning the physiologic relevance of this regulation (Murali et al, 2012).…”

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…Morphine also acts at m-opioid receptors that are expressed in the CNS (Diaz et al, 1995;Goodchild et al, 2004), where a clear correlation between physiologic effects and modulation of Ca V 2.2 channels is more difficult to establish. Although morphine is considered selective for m-opioid receptors, selective agonists of the other three members of the extended opioid receptor family (i.e., d-and k-opioid receptors, and nociceptin receptors) also functionally inhibit Ca V 2.2 channels (Gross and Macdonald, 1987;Moises et al, 1994;Motin et al, 1995;Morikawa et al, 1998;Toselli et al, 1999;Larsson et al, 2000;Yeon et al, 2004;Ruiz-Velasco et al, 2005;Evans et al, 2010). As in the case of m-opioid receptors, their activation induces analgesia in various animal models of pain (King et al, 1997;Darland et al, 1998;Field et al, 1999;Mika et al, 2001;Courteix et al, 2004;Nozaki et al, 2012).…”

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

“…Numerous studies have shown that the MOP receptor is subjected to this type of regulation by interacting with a large number of GPCRs (25,26). In the case of MOP receptor dimerization with somatostatin sst 2A (27), substance P NK1 (28), ␣ 2a -adrenergic (29), and nociceptin ORL1 (30) receptors, cross-desensitization occurs and could result from co-internalization of both partners upon activation of only one receptor or from allosteric changes in binding affinity (31). However, in the case of NPFF 2 and MOP receptors previously shown to physically interact in SH-SY5Y cells (32), the stimulation of NPFF 2 receptors does not induce the internalization of MOP receptors but, conversely, partly blocks its trafficking (32).…”