Cooperative Conformational Changes in a G-protein-coupled Receptor Dimer, the Leukotriene B4 Receptor BLT1

Mesnier, Danielle; Banères, Jean-Louis

doi:10.1074/jbc.m404941200

Cited by 67 publications

(83 citation statements)

References 35 publications

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“…First, the D71A TRH receptor mutant may be converted to an active conformation by dimerization with agonist-activated 4Ala-TRH mutant receptor, causing GRKs to phosphorylate the D71A receptor. There is precedent for this model because the agonist induces cooperative conformational changes in the leukotriene B4 receptor-BLT1 dimer (25). This model is not likely to explain our findings, however, because regulated dimerization of the TRH receptor does not initiate signaling or potentiate TRH-dependent signaling (16).…”

Section: Discussionmentioning

confidence: 79%

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Song

Jones

Hinkle

2007

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

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The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355-365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355-365 region signal normally but lack phosphorylation sites. When D71A-and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization. T he thyrotropin (ISH)-releasing hormone (TRH) receptorbelongs to the superfamily of seven transmembrane helix G protein-coupled receptors (GPCRs). TRH is a tripeptide that functions as a hormone regulating TSH and prolactin secretion. TRH receptors signal through G q and G 11 , leading to the production of inositol (1, 4, 5) triphosphate and the release of intracellular calcium. After TRH binds, the TRH receptor is rapidly desensitized (1). Desensitization of GPCRs is initiated when receptors are phosphorylated by G protein-coupled receptor kinases (GRKs) or second messenger-activated kinases. Phosphorylated receptors recruit ␤-arrestins and, as a result, become uncoupled from G proteins. Once docked on activated GPCRs, ␤-arrestins also bind to clathrin and adapter proteins, causing receptor internalization (2).A growing body of evidence suggests that many GPCRs, including the TRH receptor, form dimers or oligomers (3-6). Atomic force microscopy reveals higher order oligomers of rhodopsin (7). Receptor heterodimerization also has been shown to modulate ligand binding, signaling, and trafficking. Ligandbinding properties are altered by heterodimerization of ...

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Section: Discussionmentioning

confidence: 79%

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Song

Jones

Hinkle

2007

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

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“…Mutagenesis and cysteine cross-linking experiments involving the D 2 -dopamine receptor suggest that conformational changes occur at the dimer interface in transmembrane domain four during receptor activation and inactivation (Guo et al, 2005). In addition, agonist binding to the leukotriene B 4 receptor has been reported to induce conformational changes in the unliganded protomer of the dimer (Mesnier et al, 2004), indicating that conformational changes resulting from receptor activation are translated across the dimer interface. These results are consistent with reports of trans-activation of Gproteins following ligand binding to one protomer of the dimer Hlavackova et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

Serotonin 5-HT2C receptor homodimerization is not regulated by agonist or inverse agonist treatment

Herrick‐Davis

Grinde

Weaver

2007

European Journal of Pharmacology

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Serotonin 5-HT 2C receptors represent targets for therapeutics aimed at treating anxiety, depression, schizophrenia, and obesity. Previously, we demonstrated that 5-HT 2C receptors function as homodimers. Herein, we investigated the effect of agonist and inverse agonist treatment on the homodimer status of two naturally occurring 5-HT 2C receptor isoforms, one without basal activity (VGV) and one with constitutive activity (INI) with respect to Gα q signaling. Cyan-and yellowfluorescent proteins were used to monitor VGV and INI homodimer formation by western blot, and in living cells using bioluminescence and fluorescence resonance energy transfer (BRET and FRET). Western blots of solubilized membrane proteins revealed equal proportions of homodimeric receptor species from HEK293 cells transfected with either the VGV or INI isoform in the absence and presence of 5-HT. BRET ratios measured in HEK293 cells transfected with the VGV or INI isoform were the same and were not modulated by 5-HT. Similarly, FRET efficiencies were the same regardless of whether measured in cells expressing the VGV or INI isoform in the absence or presence of 5-HT or clozapine. The results indicate that serotonin 5-HT 2C receptors form homodimers regardless of whether they are in an inactive or active conformation and are not regulated by drug treatment.

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“…The heterodimers were then refolded and purified as described by Damian et al (9). 5HW was introduced in the receptors by biosynthetic labeling using the method described by Mesnier and Banères (17).…”

Section: Materials-ltbmentioning

confidence: 99%

G Protein Activation by the Leukotriene B4 Receptor Dimer

Damian

Mary

Martin

et al. 2008

Journal of Biological Chemistry

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There is compelling evidence that G protein-coupled receptors exist as homo-and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B 4 receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B 4 but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins. GPCRs2 are versatile biological sensors that are responsible for the majority of cellular responses to hormones and neurotransmitters as well as for the senses of sight, smell, and taste (1, 2). Signal transduction is associated with a set of changes in the tertiary structure of the receptor that are recognized by the associated intracellular partners, in particular the G proteins (3).It has been recently shown that receptor monomers can efficiently activate their G protein partners (4 -7). However, both homo-and heterodimers have been described for many GPCRs in transfected cells, although more work is needed to extend these observations to native tissues (8). The exact molecular mechanisms governing the functioning of GPCR dimers/oligomers are not clear so far. There is increasing evidence that the two protomers in a dimer are not totally equivalent. In the case of the LTB 4 receptor BLT1 homodimer, we have evidence that only one of the protomers is activated at one time (9). In the same way, only about half of the rhodopsin present in lipid nanodiscs containing two rhodopsins is available to interact with transducin, suggesting an asymmetric functioning in this case also (10). A similar explanation has been provided for the differences in G protein activation between the neurotensin receptor monomer and dimer (7). Finally, for class C receptors, several data also support a model where the receptor dimer functions in an asymmetrical way, a single heptahelical domain being activated at a time (11, 12). In the same way, only one subunit (GB2) is able to activate G proteins in the heterodimeric GABA(B) receptor (13,14).The existence of GPCR dimers has led to the concept...

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Cooperative Conformational Changes in a G-protein-coupled Receptor Dimer, the Leukotriene B4 Receptor BLT1

Cited by 67 publications

References 35 publications

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

Serotonin 5-HT2C receptor homodimerization is not regulated by agonist or inverse agonist treatment

G Protein Activation by the Leukotriene B4 Receptor Dimer

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