The Third Intracellular Loop Stabilizes the Inactive State of the Neuropeptide Y1 Receptor

Chee, Melissa J.; Mörl, Karin; Lindner, Diana; Merten, Nicole; Zamponi, Gerald W.; Light, Peter E.; Beck‐Sickinger, Annette G.; Colmers, William F.

doi:10.1074/jbc.m804671200

Cited by 35 publications

(23 citation statements)

References 69 publications

(51 reference statements)

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“…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).…”

Section: Voltage-independent Inhibition Of Ca V 2 Channelssupporting

confidence: 61%

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

Proft

Weiss

2014

Mol Pharmacol

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Neuronal voltage-gated calcium channels have evolved as one of the most important players for calcium entry into presynaptic endings responsible for the release of neurotransmitters. In turn, and to fine-tune synaptic activity and neuronal communication, numerous neurotransmitters exert a potent negative feedback over the calcium signal provided by G protein-coupled receptors. This regulation pathway of physiologic importance is also extensively exploited for therapeutic purposes, for instance in the treatment of neuropathic pain by morphine and other m-opioid receptor agonists. However, despite more than three decades of intensive research, important questions remain unsolved regarding the molecular and cellular mechanisms of direct G protein inhibition of voltage-gated calcium channels. In this study, we revisit this particular regulation and explore new considerations.

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Section: Voltage-independent Inhibition Of Ca V 2 Channelssupporting

confidence: 61%

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

Proft

Weiss

2014

Mol Pharmacol

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“…2B). The ability of dopamine analog 3 to activate WT D 2 R, as well as the ability of the covalent 3-D 2 R L94C complex to induce G protein-promoted signaling, was assessed in an inositol phosphate (IP) formation assay using HEK cells transiently transfected with a chimeric Gα qi5 protein and the WT and mutant receptor, respectively (6,23) (Fig. 2D and SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Purified WT β 2 AR or β 2 AR H93C reconstituted into recombinant HDL particles (22) was used as described (20) , and H 1 Y87C receptors was studied using IP accumulation assays as described (6,23). For activation studies with G i -coupled GPCRs, HEK 293 cells were transiently cotransfected with cDNA encoding for D 2 R L94C and the hybrid G protein Gα qi5 (Gα q protein with the last five amino acids at the C terminus replaced by the corresponding sequence of Gα i ; a gift from The J. David Gladstone Institutes) (36).…”

Section: Methodsmentioning

confidence: 99%

Covalent agonists for studying G protein-coupled receptor activation

Weichert

Kruse

Manglik

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating G s -, G i -, and G q -coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β 2 -adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. (1), and the resulting biochemical instability of the solubilized protein (2, 3). Protein crystallography, the most powerful tool for the study of GPCR structure, requires the formation of stable and conformationally homogeneous ligand-receptor complexes (4). High-affinity agonists with dissociation constants in the low to subnanomolar range and low off-rates facilitate stabilization of the protein throughout the process of expression, purification, and crystallogenesis (2); however, endogenous neurotransmitters usually show poor binding affinity. Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β 2 -adrenergic receptor (β 2 AR) (5) and M2 muscarinic receptor (6).To prevent ligand dissociation, irreversible ligation of electrophilic moieties like halomethylketones, isothiocyanates, Michael acceptors, or aziridinium groups of small-molecule ligands with a suitably positioned nucleophilic residue in the receptor has been used (7-16). However, irreversible ligands often suffer from incomplete cross-linking (15) and reduced receptor activation when covalent binding leads to loss of agonist efficacy (10, 16). Furthermore, their highly electrophilic nature and the abundance of nucleophilic groups in biological systems may lead to a low coupling selectivity (7, 8).Disulfide-based cross-linking approaches (17, 18) offer the advantage that the covalent binding of disulfide-...

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“…4D, expression of the -opioid receptor triggered only a small degree of constitutive channel inhibition, consistent with a low level of constitutive receptor activity (see also Ref. 22). In contrast, coexpression with either the ␦-opioid receptor or the -opioid receptor triggered large tonic, agonist-independent G protein modulation, as evident from the 2-fold increase in peak current amplitude following the prepulse.…”

Section: Orl1 Receptors Biochemically Interact With Members Of Thementioning

confidence: 95%

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

Evans

You

Hameed

et al. 2010

Journal of Biological Chemistry

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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...

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The Third Intracellular Loop Stabilizes the Inactive State of the Neuropeptide Y1 Receptor

Abstract: Constitutively active G-protein-coupled receptors (GPCRs

Cited by 35 publications

References 69 publications

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

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

Covalent agonists for studying G protein-coupled receptor activation

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

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