Cryptic Dimer Interface and Domain Organization of the Extracellular Region of Metabotropic Glutamate Receptor Subtype 1

doi:10.1074/jbc.M003226200

Cryptic Dimer Interface and Domain Organization of the Extracellular Region of Metabotropic Glutamate Receptor Subtype 1*

From the Departments of ^‡Molecular Biology and ^‖Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita-City, Osaka 565-0874, the ^¶Advanced Research Laboratory, Hitachi, Ltd., Hatoyama, Saitama 350-0395, the ^**Department of Anatomy, Nagoya University School of Medicine, Showa-Ku, Nagoya 466-8550, and the ^‡Department of Biological Sciences, Kyoto University Faculty of Medicine, Sakyo-Ku, Kyoto 606-8501, Japan

Abstract

Previously, we produced the whole extracellular region of metabotropic glutamate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor retained a ligand affinity comparable with that of the full-length membrane-bound receptor and formed a disulfide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1. A mutant, C140A, was a monomer under nonreduced conditions by SDS-polyacrylamide gel electrophoresis; however, C140A was eluted at the position similar to that of mGluR113, the wild type soluble receptor, by size exclusion column chromatography. Furthermore, C140A bound a ligand, [³H]quisqualate, with an affinity similar to that obtained by mGluR113. Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligands at magnitudes similar to those with wild type full-length RNA. Thus, elimination of the disulfide linkage did not perturb the dimer formation and ligand signaling, suggesting that cryptic dimer interface(s) possibly exist in mGluR1. Limited proteolysis of the whole extracellular fragment (residue 33–592) revealed two trypsin-sensitive sites, after the residues Arg¹³⁹ and Arg⁵²¹. A 15-kDa NH₂-terminal proteolytic fragment (residue 33–139) was associated with the downstream part after the digestion. Arg⁵²¹ was located before a cysteine-rich stretch preceding the transmembrane region. A new shorter soluble receptor (residue 33–522) lacking the cysteine-rich region was designed based on the protease-sensitive boundary. The purified receptor protein gave aK _d value of 58.1 ± 0.84 nm, which is compatible to a reported value of the full-length receptor. TheB _max value was 7.06 ± 0.82 nmol/mg of protein. These results indicated that the ligand-binding specificity of mGluR1 is confined to the NH₂-terminal 490-amino acid region of the mature protein.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Both authors contributed equally to this work.
↵§§ To whom correspondence should be addressed. Tel.: 81-6-6872-8214; Fax: 81-6-6872-8219; E-mail: jingami@beri.co.jp.
Published, JBC Papers in Press, June 28, 2000, DOI 10.1074/jbc.M003226200
Abbreviations:

mGluR

metabotropic glutamate receptor

CaR

calcium sensing receptor

GPCR

G protein-coupled receptor

LIVBP

leucine/isoleucine/valine-binding protein

EGS

ethylene glycol bis(succinimidylsuccinate)

CAPS

N-cyclohexyl-3-aminopropanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

mAb

monoclonal antibody

PEG

polyethylene glycol

DTT

dithiothreitol

LBD

ligand binding domain

CRD

cysteine-rich domain

bp

base pair(s)

kbp

kilobase pair(s)

GABA_B

γ-aminobutyric acid B
- Received April 14, 2000.
- Revision received June 27, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.