Kang Hatan, Lee Won Kyu, Choi Yun Hui, Vukoti Krishna Moorthy, Bang Won Gi, Yu Yeon Gyu
Life Sciences Division, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea.
Biochem Biophys Res Commun. 2005 Apr 8;329(2):684-92. doi: 10.1016/j.bbrc.2005.02.040.
The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.
5-羟色胺6型（5-HT(6)）受体是一种与刺激性G蛋白（G(S)）偶联的G蛋白偶联受体（GPCR）。为了确定5-HT(6)受体与G(S)蛋白相互作用的结构基础，我们剖析了含有5-HT(6)受体的第二个细胞内环（iL2）、第三个细胞内环（iL3）或C末端尾巴的GST融合蛋白与G(S)的α亚基（Gα(S)）之间的相互作用。通过共免疫沉淀证明了iL3与Gα(S)的直接相互作用。此外，通过表面等离子体共振测量了iL3与Gα(S)相互作用的动力学参数，确定其表观解离常数为0.9×10(-6)M。相比之下，第二个细胞内环和C末端尾巴区域对Gα(S)的亲和力可忽略不计。通过测量用5-HT(6)受体突变体转染的细胞在5-HT刺激下产生的细胞内cAMP水平，确定了iL3区域内与Gα(S)相互作用的关键残基为iL3 C末端附近保守的带正电荷残基。
