Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells

A Nomura, R Shigemoto, Y Nakamura, N Okamoto… - Cell, 1994 - cell.com
A Nomura, R Shigemoto, Y Nakamura, N Okamoto, N Mizuno, S Nakanishi
Cell, 1994cell.com
Results Characterization of mGluRG-Specific Antibody The mGluR family consists of at least
seven different subtypes that can be classified into three subgroups according to their
sequence similarities, agonist selectivities, and signal transduction mechanisms:
mGluRlImGluR5, mGluR2/mGluR3, and mGluR4/mGluRG/mGluR7 (Nakanishi, 1992;
Okamoto et al., 1994). A peptide corresponding to the 19 amino acid C-terminus unique for
mGluR6 was synthesized and used for raising a polyclonal antibody. We first tested the …
Results
Characterization of mGluRG-Specific Antibody The mGluR family consists of at least seven different subtypes that can be classified into three subgroups according to their sequence similarities, agonist selectivities, and signal transduction mechanisms: mGluRlImGluR5, mGluR2/mGluR3, and mGluR4/mGluRG/mGluR7 (Nakanishi, 1992; Okamoto et al., 1994). A peptide corresponding to the 19 amino acid C-terminus unique for mGluR6 was synthesized and used for raising a polyclonal antibody. We first tested the specificity of the polyclonal antibody obtained by examining its immunoreactivity with mGluR6 and the other L-APCsensitive mGluR4 and mGluR7 subtypes. On immunoblot analysis, two immunoreactive bands with estimated molecular masses of 110 kd and 92 kd, together with those of higher molecular mass components of 180-200 kd, were detected in membrane fractions of mGluRG-expressing CHO cells (Figure 1, lane 3) but not in those of either CHO cells transfected with the vector DNA alone (lane 1) or mGluRC or mGluR7-expressing CHO cells (lanes 2 and 4). All of these immunoreactive bands disappeared by antibody preabsorption with the synthetic peptide antigen (Figure 1, lane 5). The estimated molecular masses of 110 kd and 92 kd for mGluR6 were slightly different from that of the cloned mGluR6 (95 kd), and this difference may result from posttranslational modifications such as glycosylation and proteolytic cleavage in cultured cells (Hayashi et al., 1993; Shigemoto et al., 1993). The higher molecular mass components probably represent
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