NMR structural study of the intracellular loop 3 of the serotonin 5-HT1A receptor and its interaction with calmodulin

The serotonin (5-HT1A) receptor, a G-protein-coupled receptor (GPCR), plays important roles in serotonergic signaling in the central nervous system. The third intracellular loop (ICL3) of the 5-HT1A receptor has been shown to be important for the regulation of this receptor through interactions with proteins such as G-proteins and calmodulin. In this study, the ICL3 of 5-HT1A receptor was expressed in E. coli and purified. Gel filtration and mass spectrometry were used to confirm the molecular weight of the purified ICL3. Secondary structure analysis using circular dichroism (CD) demonstrated the presence of alpha-helical structures. Backbone assignment of ICL3 was achieved using three-dimensional experiments. A chemical shift index and Talos+ analysis showed that residues E326 to R339 form alpha-helical structure. Residues G256 to 5269 of ICL3 were shown to be a novel region that has a molecular interaction with calmodulin in titration assays. Peptide derived from the ICL3 containing residues from G256 to S269 also showed molecular interaction with calmodulin. (C) 2011 Elsevier B.V. All rights reserved.