Premium
Mutations in the i3N loop calmodulin (CaM)‐binding domain of 5‐HT 1A receptor attenuate ERK phosphorylation in CHO‐K1 cells
Author(s) -
Coaxum Sonya Denise,
Raymond John R.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.728.8
Subject(s) - chinese hamster ovary cell , receptor , mapk/erk pathway , microbiology and biotechnology , enzyme linked receptor , transfection , biotinylation , 5 ht5a receptor , phosphorylation , chemistry , biology , biochemistry , gene
Our laboratory previously has shown that calmodulin (CaM) binds to two sites in the 5‐HT 1A receptor i3 loop; however, the functional significance of the interaction of CaM with the 5‐HT 1A receptor is not clear. Because ERK activation is one of the primary signals of the G‐protein coupled 5‐HT 1A receptor, we wanted to determine if a receptor deficient in CaM binding has altered ERK activation. We constructed a double point mutation (R223E/K232E) in the 5‐HT 1A receptor, and demonstrated that it was well‐expressed after transfection into CHO cells. It binds normally to two agonists (5‐HT and 8‐OH‐DPAT) and maintains intact coupling of the receptor to G‐proteins as demonstrated by GTP‐sensitive high affinity binding. Dot‐blots with biotinylated CaM‐enzyme‐linked avidin system showed that CaM binds to the wild‐type 5‐HT 1A peptide in the presence of Ca 2+ ; in contrast, binding of CaM to [R223E/K232E]5‐HT 1A i3N peptide was greatly attenuated, suggesting that the affinity of the mutated 5‐HT 1A peptide for CaM was markedly reduced. We stably transfected CHO‐K1 cells with the wild‐type 5‐HT 1A receptor or [R223E/K232E]5‐HT 1A receptor construct, and measured ERK phosphorylation over time. ERK phosphorylation peaked between 5 and 10 minutes for the wild‐type 5‐HT 1A receptor, and peaked at 5 minutes for the mutated [R223E/K232E]5‐HT 1A receptor. Interestingly, ERK phosphorylation was decreased dramatically in CHO‐K1 cells transfected with the [R223E/K232E]5‐HT 1A receptor construct when compared with the wild‐type 5‐HT 1A receptor. These results strongly support a critical role for direct CaM binding to the 5‐HT 1A receptor for effective ERK phosphorylation.