Crystal Structure of Squid Rhodopsin with Intracellularly Extended Cytoplasmic Region

Shimamura, Tatsuro; Hiraki, Kenji; Takahashi, Naoko; Hori, Tetsuya; Ago, Hideo; Masuda, Koichi; Takio, Koji; Ishiguro, Masaji; Miyano, Masashi

doi:10.1074/jbc.c800040200

Cited by 123 publications

(132 citation statements)

References 32 publications

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“…The helix 8 region of GPCRs forms an amphipathic helix that lies parallel to the plasma membrane and is anchored by palmitoylated cysteine groups (Palczewski et al, 2000;Shimamura et al, 2008). Helix 8 is believed to move upon receptor activation.…”

Section: Discussionmentioning

confidence: 99%

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Gehret¹,

Jones²,

Tran³

et al. 2009

Mol Pharmacol

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The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X) 5-6 F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the ␤2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.The type 1 TRH receptor is a GPCR expressed in the anterior pituitary gland. In response to TRH, the receptor activates G q/11 , which in turn stimulates phospholipase C. The resulting increases in inositol trisphosphate and diacylglycerol cause rapid release of intracellular calcium and activation of protein kinase C. Like many GPCRs, the TRH receptor contains a canonical NPXXY at the end of transmembrane 7 in a NPXXY(X) 5-6 F(R/K) motif (Mirzadegan et al., 2003;Okuno et al., 2005;Swift et al., 2006) and is predicted to form an amphipathic eighth helix with a positively charged face ending at a downstream pair of cysteine residues, where palmitoylation occurs (Du et al., 2005).When TRH binds, the receptor undergoes rapid and quantitative phosphorylation at multiple sites in the cytoplasmic tail Hinkle, 2005, 2008;Jones et al., 2007). Agonist-stimulated phosphorylation of the receptor drives arrestin binding and arrestin-dependent receptor desensitization and internalization. The receptor tail is not detectably phosphorylated before activation.Agonist-dependent phosphorylation of GPCRs is usually carried out by either GRKs that recognize the activated state of the receptor or downstream kinases that are activated by receptor signaling. Th...

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Section: Discussionmentioning

confidence: 99%

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Gehret¹,

Jones²,

Tran³

et al. 2009

Mol Pharmacol

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“…Crystal structures of G proteins have been obtained in both active and inactive states (4)(5)(6), and structures of bovine rhodopsin (7)(8)(9)(10)(11)(12), squid rhodopsin (13,14), bovine opsin (15,16), the human ␤ 2 AR (17)(18)(19), the turkey ␤ 1 AR (20), and the adenosine A2a receptor (21) have been reported. However, relatively little is known about the active-state GPCR-G protein complex.…”

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confidence: 99%

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

Yao

Ruiz

Whorton

et al. 2009

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

217

203

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G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the ␤2 adrenergic receptor (␤2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The ␤2AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist-and Gs-induced conformational changes. In the absence of an agonist, the ␤2AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the ␤2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the ␤ 2 AR and the structural basis for ligand efficacy.adrenergic ͉ constitutive activity ͉ receptor ͉ conformation ͉ inverse agonist

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“…The crystal structures of bovine and squid rhodopsins 16,29,30) suggested that the Arg3.50 in the inactive state forms salt bond with the adjacent Glu or Asp3.49 residue of TMH III. Upon mutation of the adjacent Glu or Asp3.49, the arginine3.50 residue will be released from the electronic constraint leading to a conformational change (Fig.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Inverse Agonist Action of Sarpogrelate at the Constitutively Active Mutant of Human 5-HT<sub>2A</sub> Receptor Revealed by Molecular Modeling

Hossain

Muntasir

Ishiguro

et al. 2012

Biological & Pharmaceutical Bulletin

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We previously reported that sarpogrelate, a selective 5-HT 2A antagonist, showed a potent inverse agonist activity to constitutively active mutant (C322K) of human 5-HT 2A receptor (5-HT 2A R). However, it remains to be unknown about the actual mechanism of this mutant for its constitutive activation as well as inverse agonist activity of sarpogrelate. Our model shows that mutation (C322K) of 5-HT 2A R causes electronic repulsion between positively charged Arg173(3.50) and Lys322(6.34) residues resulting outward movement of the C-terminus of transmembrane helix (TMH) III. This motion of TMH III leads to a partially active structure of the receptor, which may be a key step in receptor activation. The structural model of the partially active receptor also indicates that the binding of sarpogrelate to the constitutively active receptor causes an inward swing of TMH III to an inactive receptor structure. Therefore, the present study may suggest that the electronic repulsion causing outward movement of the C-terminus of TMH III may be the key step for constitutive activation of mutant C322K of 5-HT 2A R and the inward movement of TMH III causes the inverse agonist activity of sarpogrelate.Key words 5-HT 2A receptor; mutation; constitutive activity; molecular modeling; electronic repulsion It has been established that many G-protein coupled receptors (GPCRs) can exist in a constitutively active form in the absence of agonist.1-3) Certain molecules could act as inverse agonists and reduce the levels of constitutive activity and functional cellular responses. [3][4][5][6] Inverse agonism is very common among GPCR antagonists.7) It has become apparent over the past few years that a number of compounds that had been thought to be neutral antagonists at GPCRs possess negative efficacy and have been termed inverse agonists. This was first shown for μ-opiate and α 2 -adrenergic receptors 1,8) but has subsequently been extended to many GPCRs. 9,10) Considering the therapeutic implication, it is suggested that all new antagonists should be routinely tested for their potential inverse agonistic activity in future drug development programs. 11,12) This concept is of particular importance for the 5-HT 2A receptor (5-HT 2A R) where the drugs used to treat cardiovascular diseases had been assumed to be antagonists at this receptor. It has, however, been shown using potentiation of basal inositol phosphate activity 13) that all of the drugs tested were inverse agonists and there was a good correlation between inverse agonist potency and ligand binding affinity. In the previous study, we demonstrated that sarpogrelate, a selective 5-HT 2A antagonist, had been shown to be a potent inverse agonist at the constitutive active human 5-HT 2A R. 14) Patients are treated chronically with this drug and the effects of an inverse agonist on the activity of 5-HT 2A R may be quite different to those of an antagonist. Owing to the important therapeutic activity (i.e. to treat ischemic diseases) of sarpogrelate, it is very important to understand t...

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Crystal Structure of Squid Rhodopsin with Intracellularly Extended Cytoplasmic Region

Cited by 123 publications

References 32 publications

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

Mechanism of Inverse Agonist Action of Sarpogrelate at the Constitutively Active Mutant of Human 5-HT<sub>2A</sub> Receptor Revealed by Molecular Modeling

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