Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization

Carmelo Romano, Judith Kelvin Miller, Krzysztof Hyrc, Seta Dikranian, Steven Mennerick, Yutaka Takeuchi, Mark P. Goldberg, Karen L. O'Malley

Research output: Contribution to journalArticle

113 Citations (Scopus)

Abstract

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity simiiar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

Original languageEnglish (US)
Pages (from-to)46-53
Number of pages8
JournalMolecular Pharmacology
Volume59
Issue number1
StatePublished - 2001

Fingerprint

Metabotropic Glutamate Receptors
Dimerization
Oocytes
Cysteine
Quisqualic Acid
Fura-2
Xenopus laevis
G-Protein-Coupled Receptors
Disulfides
Glutamic Acid
Calcium
Kidney

ASJC Scopus subject areas

  • Pharmacology

Cite this

Romano, C., Miller, J. K., Hyrc, K., Dikranian, S., Mennerick, S., Takeuchi, Y., ... O'Malley, K. L. (2001). Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization. Molecular Pharmacology, 59(1), 46-53.

Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization. / Romano, Carmelo; Miller, Judith Kelvin; Hyrc, Krzysztof; Dikranian, Seta; Mennerick, Steven; Takeuchi, Yutaka; Goldberg, Mark P.; O'Malley, Karen L.

In: Molecular Pharmacology, Vol. 59, No. 1, 2001, p. 46-53.

Research output: Contribution to journalArticle

Romano, C, Miller, JK, Hyrc, K, Dikranian, S, Mennerick, S, Takeuchi, Y, Goldberg, MP & O'Malley, KL 2001, 'Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization', Molecular Pharmacology, vol. 59, no. 1, pp. 46-53.
Romano C, Miller JK, Hyrc K, Dikranian S, Mennerick S, Takeuchi Y et al. Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization. Molecular Pharmacology. 2001;59(1):46-53.
Romano, Carmelo ; Miller, Judith Kelvin ; Hyrc, Krzysztof ; Dikranian, Seta ; Mennerick, Steven ; Takeuchi, Yutaka ; Goldberg, Mark P. ; O'Malley, Karen L. / Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization. In: Molecular Pharmacology. 2001 ; Vol. 59, No. 1. pp. 46-53.
@article{33ed286034b84dbcb66402e1c2dce4f1,
title = "Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization",
abstract = "Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity simiiar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.",
author = "Carmelo Romano and Miller, {Judith Kelvin} and Krzysztof Hyrc and Seta Dikranian and Steven Mennerick and Yutaka Takeuchi and Goldberg, {Mark P.} and O'Malley, {Karen L.}",
year = "2001",
language = "English (US)",
volume = "59",
pages = "46--53",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "1",

}

TY - JOUR

T1 - Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization

AU - Romano, Carmelo

AU - Miller, Judith Kelvin

AU - Hyrc, Krzysztof

AU - Dikranian, Seta

AU - Mennerick, Steven

AU - Takeuchi, Yutaka

AU - Goldberg, Mark P.

AU - O'Malley, Karen L.

PY - 2001

Y1 - 2001

N2 - Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity simiiar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

AB - Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity simiiar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

UR - http://www.scopus.com/inward/record.url?scp=0035164277&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035164277&partnerID=8YFLogxK

M3 - Article

VL - 59

SP - 46

EP - 53

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 1

ER -