Conversion of the human 5-HT1Dβ serotonin receptor to the rat 5-HT1b ligand-binding phenotype by Thr355 ASN site directed mutagenesis

Metcalf, Mark A.; McGuffin, Robert W.; Hamblin, Mark W.

doi:10.1016/0006-2952(92)90092-w

Cited by 72 publications

(27 citation statements)

References 12 publications

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“…As expected based on the literature (16,(18)(19)(20), substitution of AsnVII:06 with either His or Cys impaired the affinity for pindolol and thereby eliminated the binding of 125 I-labeled pindolol (data not shown) and also impaired the ability of pindolol to stimulate cAMP production ( Table 1). Despite the fact that only a single potential metal-ion binding residue had been introduced in the CysVII:06 mutant, Zn 2ϩ in the form of ZnCl 2 was able to stimulate cAMP production with an EC 50 of 91 M ( Fig.…”

Section: Resultssupporting

confidence: 87%

“…Asn-312 (AsnVII:06) was chosen as a starting point for metal-ion site engineering in the ␤ 2 -adrenergic receptor because it previously has been characterized as an interaction site for partial agonists such as pindolol (16,(18)(19)(20). Substitution of AsnVII:06 with either His or Cys did not affect the basal signaling activity of the receptor.…”

Section: Resultsmentioning

confidence: 99%

“…† In monoamine receptors a well-characterized interaction site for the amine group of the ligands is AspIII:08 being located one helical turn below GluIII:04 of rhodopsin (17). In TM-VII of the catecholamine and 5-hydroxytryptamine (5HT) receptors residue VII:06 has consistently been identified as an interaction site for partial agonists as well as antagonists as based on mutational studies giving even up to 1,000-fold gain of affinity (16,(18)(19)(20). Residue VII:06 is located one helical turn above LysVII:10 of rhodopsin and is conceivably facing rather directly toward TM-III (Fig.…”

mentioning

confidence: 99%

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Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor

Elling¹,

Thirstrup²,

Holst³

et al. 1999

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

109

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Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the ␤2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu 2؉ , and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structureactivity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will. R hodopsin-like G protein-coupled receptors, which constitute the largest superfamily of proteins in our organism, are believed to have a common seven helical transmembrane (7TM) fold and conceivably a common, still elusive molecular mechanism of activation (1). Cryoelectron microscopy of rhodopsin has provided the basis for a generally accepted model for the overall structure of 7TM receptors (2-5). However, their detailed three-dimensional structure and the conformational changes leading to their activation is still rather ill-defined (6-8).Previously metal-ion site engineering has been employed for structural and functional analysis of 7TM receptors (9-12). Originally, the binding site for a nonpeptide antagonist was exchanged with a high affinity, tridentate Zn 2ϩ binding site located between two helices in the NK 1 tachykinin receptor (9). This site could be transferred as a zinc switch without loss of metal-ion affinity to the distantly related -opioid receptor, indicating that the overall structure within the family of rhodopsin-like 7TM receptors is well conserved (10). Interhelical bidentate zinc sites subsequently have been used to probe helix-helix interactions and, for example to prove that the seven helical bundle is arranged in an anti-clock-wise arrangement as viewed from outside the cell (11). However, as yet all metal-ion sites created both by us and others have been antagonistic (9-13). Such sites are inte...

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor

Elling¹,

Thirstrup²,

Holst³

et al. 1999

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

109

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“…A slight increase in antagonist affinity was noted for the chimera E4. The fourth extracellular domain is directly followed by the seventh transmembrane helix which has been shown to participate in binding to antagonists for the ␣ 2 -adrenergic (21) and the 5-HT1D␤ serotonin receptor (22).…”

Section: The Oxytocin Antagonist Binding Site Is Formed By the Transmmentioning

confidence: 99%

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Postina

Kojro

Fahrenholz

1996

Journal of Biological Chemistry

119

102

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The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V 2 vasopressin receptor, chimeric "gain in function" V 2 /OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. Oxytocin belongs to the family of neurohypophyseal nonapeptide hormones, which are characterized by a cyclic hexapeptidic part with a disulfide bridge between Cys 1 and Cys 6 and a C-terminal linear tripeptidic extension (Fig. 1). Its major physiological role is to induce contraction of uterine smooth muscle and mammary myoepithelium. As pregnancy nears term, uterine smooth muscle exhibits enhanced sensitivity to oxytocin (1). Oxytocin is widely used to induce labor, and conversely, oxytocin antagonists are being evaluated for the treatment of preterm labor (2, 3). Furthermore, OT 1 has been shown to play an important role in reproduction biology by influencing sexual behavior and response, as well as the formation of social bonds (4). These oxytocinergic effects are mediated by a specific cell surface receptor, the OT receptor which is also found in other organs such as brain (5) and ovary (6). Cloning of the oxytocin receptor from five mammalian species has shown that it belongs to the G protein-coupled receptor family with seven transmembrane helices (7-11). Photoaffinity labeling studies with a photoreactive OT antagonist identified the oxytocin receptor as a glycoprotein with an apparent molecular weight between 68,000 and 80,000 (12). Functional domains, especially the topography of the ligand-binding site, are presently unknown.The aim of our investigations was to determine domains of the OT receptor that are involved in high-affinity binding of both agonists and antagonists. Since all known OT receptors share almost identical primary structure and bind OT with high affinity, it can be assumed that alterations of this naturally optimized binding site will result in a loss of high-affinity oxytocin binding. This effect can be caused either by disturbances of specific receptor-ligand interactions but also by changes in the overall receptor conformation. Therefore, we decided to produce "gain in function" receptors, to identify OT receptor domains that contribute to high-affinity OT and oxytocin antagonist binding.As starting protein for the identification of oxytocin binding domains, we used the related V 2 vasopressin receptor. This receptor mediates the antidiuretic action of [8-arginine]vasopressin in kidney. It shows 40% overall sequence identity to the oxytocin receptor with the highest sequence similarity in the transmembrane regions and the extracellular loops. Although vasopressin and oxytocin differ only at position 3 of the cyclic peptide part and position 8 of the C-terminal tripeptide amide, the V 2 vasopressin receptor dis...

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“…The known pharmacological differences between rodent and human 5-HTiB receptors are a likely result of a single amino acid difference be tween the rodent 5-HTib receptor and all oth er 5-HT1B and 5-HTiD receptors. Specifically, the molecular basis for the pharmacological differences between the highly homologous human and rodent 5-HTib receptor genes has been localized to the seventh transmembrane segment of the 5-HTib receptor [16,24,25]. Therefore, these data indicate that a single amino acid difference can elicit a significant change in the pharmacological profile of a receptor without altering the affinity of the endogenous ligand.…”

Section: Discussionmentioning

confidence: 99%

Pharmacological Differentiation of Human 5-HT<sub>1B</sub> and 5-HT<sub>1D</sub> Receptors

Peroutka¹

1994

Biological Signals

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Human 5-hydroxytryptamine_1B (5-HT_1B; also designated 5-HT_1D_β) receptors and 5-HT_1D (also designated 5-HT_1Dα) receptors are distinct molecular entities which mediate serotonergic neurotransmission. The often confusing variability in nomenclature for these two G protein-coupled receptors derives from a preliminary observation that these receptors display nearly indistinguishable pharmacological properties. The present study analyzed a series of 21 drugs, 8 of which were found to be at least an order of magnitude more potent at 5-HT_1D than 5-HT_1B receptors. These data provide pharmacological evidence that human 5-HT_1B and 5-HT_1D receptors are distinct molecular entities which represent independent targets for future drug development. Furthermore, these data indicate that a number of selective pharmacological agents already exist and can be used to analyze the functional roles of 5-HT_1B and 5-HT_1D receptors.

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Conversion of the human 5-HT1Dβ serotonin receptor to the rat 5-HT1b ligand-binding phenotype by Thr355 ASN site directed mutagenesis

Cited by 72 publications

References 12 publications

Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor

Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Pharmacological Differentiation of Human 5-HT<sub>1B</sub> and 5-HT<sub>1D</sub> Receptors

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Conversion of the human 5-HT1Dβ serotonin receptor to the rat 5-HT1b ligand-binding phenotype by Thr355 ASN site directed mutagenesis

Cited by 72 publications

References 12 publications

Conversion of agonist site to metal-ion chelator site in the β 2 -adrenergic receptor

Conversion of agonist site to metal-ion chelator site in the β 2 -adrenergic receptor

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Pharmacological Differentiation of Human 5-HT<sub>1B</sub> and 5-HT<sub>1D</sub> Receptors

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Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor

Conversion of agonist site to metal-ion chelator site in the β ₂ -adrenergic receptor