Cryptic dimer interface and domain organization of the extracellular region of metabotropic glutamate receptor subtype 1

Previously, we produced the whole extracellular region of metabotropic glut amate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor r etained a ligand affinity comparable with that of the full-length membrane- bound receptor and formed a disulfide-linked dimer, Here, we have identifie d a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1, A mut ant, 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, [H-3]quisqualate, with a n affinity similar to that obtained by mGluR113, Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligand s at magnitudes similar to those with wild type full-length RNA. Thus, elim ination of the disulfide linkage did not perturb the dimer formation and li gand signaling, suggesting that cryptic dimer interface(s) possibly exist i n mGluR1, Limited proteolysis of the whole extracellular fragment (residue 33-592) revealed two trypsin-sensitive sites, after the residues Arg(139) a nd Arg(521). A 15-kDa NH2-terminal proteolytic fragment (residue 33-139) wa s associated with the downstream part after the digestion. Arg(521) was loc ated before a cysteine-rich stretch preceding the transmembrane region. A n ew shorter soluble receptor (residue 33-522) lacking the cysteine-rich regi on was designed based on the protease-sensitive boundary. The purified rece ptor protein gave a K-d value of 58.1 +/- 0.84 nM, which is compatible to a reported value of the full-length receptor. The B-max value was 7.06 +/- 0 .82 nmol/mg of protein. These results indicated that the ligand-binding spe cificity of mGluR1 is confined to the NH2-terminal 490-amino acid region of the mature protein.