Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

Pellissier, Lucie P.; Sallander, Jessica; Campillo, Mercedes; Gaven, Florence; Queffeulou, Emilie; Pillot, Marion; Dumuis, Aline; Claeysen, Sylvie; Bockaert, Joël; Pardo, Leonardo

doi:10.1124/mol.108.053686

Cited by 53 publications

(67 citation statements)

References 40 publications

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“…6). Similarly, the high constitutive activity of the 5HT-4 receptor is eliminated by Ala substitution of Trp-VI:13 (15,42). However, this is not the case for the CB1 receptor, in which the constitutive activity basically is unaffected by Ala substitution of Trp-VI:13 (43) and in the B2 bradykinin receptor, where Trp-VI:13 also only plays a subtle role in controlling the balance between the active and inactive state of the receptor (44).…”

Section: Discussionmentioning

confidence: 84%

A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors

Holst

Nygaard

Valentin-Hansen

et al. 2010

Journal of Biological Chemistry

131

111

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The conserved tryptophan in position 13 of TM-VI (Trp-VI:13 or Trp-6.48) of the CWXP motif located at the bottom of the main ligand-binding pocket in TM-VI is believed to function as a rotameric microswitch in the activation process of seventransmembrane (7TM) receptors. Molecular dynamics simulations in rhodopsin demonstrated that rotation around the chi1 torsion angle of Trp-VI:13 brings its side chain close to the equally highly conserved Phe-V:13 (Phe-5.47) in TM-V. In the ghrelin receptor, engineering of high affinity metal-ion sites between these positions confirmed their close spatial proximity. Mutational analysis was performed in the ghrelin receptor with multiple substitutions and with Ala substitutions in GPR119, GPR39, and the ␤ 2 -adrenergic receptor as well as the NK1 receptor. In all of these cases, it was found that mutation of the Trp-VI:13 rotameric switch itself eliminated the constitutive signaling and strongly impaired agonist-induced signaling without affecting agonist affinity and potency. Ala substitution of Phe-V:13, the presumed interaction partner for Trp-VI:13, also in all cases impaired both the constitutive and the agonist-induced receptor signaling, but not to the same degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency. In a proposed active receptor conformation generated by molecular simulations, where the extracellular segment of TM-VI is tilted inwards in the main ligand-binding pocket, Trp-VI:13 could rotate into a position where it obtained an ideal aromatic-aromatic interaction with Phe-V: 13. It is concluded that Phe-V:13 can serve as an aromatic lock for the proposed active conformation of the Trp-VI:13 rotameric switch, being involved in the global movement of TM-V and TM-VI in 7TM receptor activation.Despite the fact that the large superfamily of 7TM 3 or G protein-coupled receptors are activated by agonists of incredibly different chemical nature, it is believed that they nevertheless all share a common molecular activation mechanism (1-3). A series of biochemical and biophysical studies indicate that receptor activation is associated with relatively large overall changes in the arrangement of the seven-helical bundle of transmembrane segments (4, 5). This notion has been gathered in a unifying "global toggle switch" activation model describing how in particular TM-VI performs a "vertical" see-saw movement around a pivot in the middle of the membrane during activation (2). Thus, the extracellular segment of TM-VI is supposed to tilt into the main ligand-binding pocket, whereas the intracellular segment tilts outward, away from the receptor center and thereby allows binding of the active form of the G protein. However, changes in the relative conformation of TM-V and -VII are also supposed to be important parts of the activation process in which the conserved proline residues in the middle of the transmembrane segments are involved, as in TM-VI (2, 6). Recently, the x-ray structure of opsin in complex with ...

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Section: Discussionmentioning

confidence: 84%

A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors

Holst

Nygaard

Valentin-Hansen

et al. 2010

Journal of Biological Chemistry

131

111

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“…Much less is known about the consequences of dimerization on receptor signaling efficiency. Here, we have addressed this question by using 5-HT 4 R as a class A GPCR model and by taking advantage of a large variety of mutants, which bind or do not bind to agonists, couple or do not couple to G protein, and can be studied in cellular contexts as well as in vitro (34,35,40,41).…”

Section: Discussionmentioning

confidence: 99%

G Protein Activation by Serotonin Type 4 Receptor Dimers

Pellissier

Barthet

Gaven

et al. 2011

Journal of Biological Chemistry

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The discovery that class C G protein-coupled receptors (GPCRs) function as obligatory dimeric entities has generated major interest in GPCR oligomerization. Oligomerization now appears to be a common feature among all GPCR classes. However, the functional significance of this process remains unclear because, in vitro, some monomeric GPCRs, such as rhodopsin and ␤ 2 -adrenergic receptors, activate G proteins. By using wild type and mutant serotonin type 4 receptors (5-HT 4 Rs) (including a 5-HT 4 -RASSL) expressed in COS-7 cells as models of class A GPCRs, we show that activation of one protomer in a dimer was sufficient to stimulate G proteins. However, coupling efficiency was 2 times higher when both protomers were activated. Expression of combinations of 5-HT 4 , in which both protomers were able to bind to agonists but only one could couple to G proteins, suggested that upon agonist occupancy, protomers did not independently couple to G proteins but rather that only one G protein was activated. Coupling of a single heterotrimeric G s protein to a receptor dimer was further confirmed in vitro, using the purified recombinant WT RASSL 5-HT 4 R obligatory heterodimer. These results, together with previous findings, demonstrate that, differently from class C GPCR dimers, class A GPCR dimers have pleiotropic activation mechanisms. G protein-coupled receptors (GPCRs)2 are key players in cell-cell communication. They transduce a wide range of extracellular signals, such as light, odors, hormones, or neurotransmitters into appropriated cellular responses (1, 2). Signal transduction occurs via conformational changes of the ligandactivated GPCRs, leading to activation of G proteins and their downstream signaling pathways (3).GPCRs have been considered for a long time as monomeric proteins, and the paradigm of "one ligand/one receptor/one G protein" was the driving principle (1). However, a growing number of studies revealed dimerization/oligomerization of GPCRs (4 -8) mostly in heterologous cells (homo-or heterodimers) but also in native tissues or in vivo (dimers) (9 -13). In line with these observations, a recent report described crystal structures of the chemokine receptor CXCR4 that are consistent with the formation of homodimers (14).A relatively accepted model proposes that only one protomer in a dimer is fully activated, even when both binding sites are occupied (15)(16)(17)(18)(19). The activated protomer interacts with the G␣ subunit to accelerate GDP/GTP exchange. This is compatible with recent data showing that a rhodopsin monomer (or a ␤ 2 -adrenergic receptor monomer) is sufficient to activate its cognate G protein after purification and reconstitution in a phospholipid bilayer (20, 21). However, some observations indicate that the active state of a GPCR dimer is asymmetric (22, 23) and that conformational switches occur between protomers (24, 25). Moreover, occupation of the second protomer of a dimer is probably not "silent" because it can either favor (26) or reduce (22) coupling efficiency. It has also ...

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“…The observation of differential signaling has supported the notion that molecules produce ligandspecific conformations in the cell membrane (Palanche et al, 2001; reviewed in Kenakin, 2001Kenakin, , 2002aKenakin, ,b, 2003. In fact, the ability of ligands to stabilize different con-7TM RECEPTORS AS SHAPESHIFTING PROTEINS formations of 7TMRs has been confirmed directly with a number of technological approaches, including fluorescent probes on receptors (Gether et al, 1995;Ghanouni et al, 2001;Kobilka and Gether, 2002), plasmonwaveguide resonance spectroscopy (Hruby and Tollin, 2007;Georgieva et al, 2008), fluorescence resonance energy transfer studies (Vilardaga et al, 2003(Vilardaga et al, , 2005Swaminath et al, 2004Swaminath et al, , 2005Granier et al, 2007;Lohse et al, 2008;Zü rn et al, 2009), bioluminescence resonance energy transfer studies (Galandrin et al, 2008;Lohse et al, 2008), circular dichroism (Banères et al, 2005), X-ray crystallography (Okada and Palczewski, 2001), antibody binding (Tutor et al, 2007), site-directed mutagenesis and molecule modeling (Pellissier et al, 2009), and kinetic studies (Swaminath et al, 2004). As mentioned previously, indirect cytosolic signaling probes have been used for a number of years to detect ligand-specific receptor conformations.…”

Section: Operational Mechanisms Of Functional Selectivity a Historimentioning

confidence: 99%

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

2010

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Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

Cited by 53 publications

References 40 publications

A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors

A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors

G Protein Activation by Serotonin Type 4 Receptor Dimers

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

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