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

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 disul-fide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extra-cellular 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]quis-qualate, 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 float 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 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 specificity of mGluR1 is confined to the NH2-terminal 490-amino acid region of the mature protein.