A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

Laura Storjohann; Birgitte Holst; Thue W Schwartz

doi:10.1021/bi8005016

A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

Laura Storjohann, Birgitte Holst, Thue W Schwartz

Institut for Neurovidenskab

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

27 Citationer (Scopus)

Abstract

Udgivelsesdato: 2008-Sep-2

Originalsprog	Engelsk
Tidsskrift	Biochemistry
Vol/bind	47
Udgave nummer	35
Sider (fra-til)	9198-207
Antal sider	9
ISSN	0006-2960
DOI	https://doi.org/10.1021/bi8005016
Status	Udgivet - 2008

Bibliografisk note

Keywords: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Cells, Cultured; Cysteine; Disulfides; Humans; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Receptors, G-Protein-Coupled; Sequence Alignment

Adgang til dokumentet

10.1021/bi8005016

Citationsformater

@article{11c47890f2f911ddbf70000ea68e967b,

title = "A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39",

abstract = "A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains. By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge connecting Cys (11) in the N-terminal domain with Cys (191) in ECL-2. Disruption of the conserved disulfide bond by mutagenesis greatly reduced the level of cell surface expression and eliminated agonist-induced increases in inositol phosphate production but surprisingly enhanced constitutive signaling. Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no effect on the receptor already lacking the second disulfide bridge and already displaying a high Zn (2+) potency. We conclude that the second disulfide bridge, which according to the beta2-adrenergic structure will form a covalent link across the entrance to the main ligand binding pocket, serves to dampen GPR39 activation. We suggest that formation of extra disulfide bridges may be an important general mechanism for regulating the activity of 7TM receptors.",

author = "Laura Storjohann and Birgitte Holst and Schwartz, {Thue W}",

note = "Keywords: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Cells, Cultured; Cysteine; Disulfides; Humans; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Receptors, G-Protein-Coupled; Sequence Alignment",

year = "2008",

doi = "10.1021/bi8005016",

language = "English",

volume = "47",

pages = "9198--207",

journal = "Biochemistry",

issn = "0006-2960",

publisher = "American Chemical Society",

number = "35",

}

TY - JOUR

T1 - A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39

AU - Storjohann, Laura

AU - Holst, Birgitte

AU - Schwartz, Thue W

N1 - Keywords: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Cells, Cultured; Cysteine; Disulfides; Humans; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Receptors, G-Protein-Coupled; Sequence Alignment

PY - 2008

Y1 - 2008

N2 - A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains. By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge connecting Cys (11) in the N-terminal domain with Cys (191) in ECL-2. Disruption of the conserved disulfide bond by mutagenesis greatly reduced the level of cell surface expression and eliminated agonist-induced increases in inositol phosphate production but surprisingly enhanced constitutive signaling. Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no effect on the receptor already lacking the second disulfide bridge and already displaying a high Zn (2+) potency. We conclude that the second disulfide bridge, which according to the beta2-adrenergic structure will form a covalent link across the entrance to the main ligand binding pocket, serves to dampen GPR39 activation. We suggest that formation of extra disulfide bridges may be an important general mechanism for regulating the activity of 7TM receptors.

AB - A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains. By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge connecting Cys (11) in the N-terminal domain with Cys (191) in ECL-2. Disruption of the conserved disulfide bond by mutagenesis greatly reduced the level of cell surface expression and eliminated agonist-induced increases in inositol phosphate production but surprisingly enhanced constitutive signaling. Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no effect on the receptor already lacking the second disulfide bridge and already displaying a high Zn (2+) potency. We conclude that the second disulfide bridge, which according to the beta2-adrenergic structure will form a covalent link across the entrance to the main ligand binding pocket, serves to dampen GPR39 activation. We suggest that formation of extra disulfide bridges may be an important general mechanism for regulating the activity of 7TM receptors.

U2 - 10.1021/bi8005016

DO - 10.1021/bi8005016

M3 - Journal article

C2 - 18693759

VL - 47

SP - 9198

EP - 9207

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 35

ER -