tailieunhanh - Báo cáo khoa học: C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

We have investigated the existence of a precoupled form of the distal C-ter-minal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production lev-els obtained in our expression system were double the values reported in the literature. | ễFEBS Journal C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity Chandramouli Reddy Chillakuri Christoph Reinhart and Hartmut Michel Department of Molecular Membrane Biology Max-Planck-Institute for Biophysics Frankfurt Main Germany Keywords cannabinoid receptor G protein coupled receptor G proteins membrane proteins signal transduction Correspondence H. Michel Department of Molecular Membrane Biology Max-Planck-Institute for Biophysics Max-von-Laue D-60438 Frankfurt Main Germany Fax 49 69 6303 1002 Tel 49 69 6303 1001 E-mail . Received 23 July 2007 revised 15 September 2007 accepted 8 October 2007 doi We have investigated the existence of a precoupled form of the distal C-ter-minal truncated cannabinoid receptor 1 CB1-417 and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization as was predicted in the literature. Using fluorescence resonance energy transfer we found that CB1-417 and Gai1p1y2 proteins were colocalized in the cells. GTPyS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Gai1 protein in the absence of agonists. A CB1-specific antagonist SR 141716A inhibited this constitutive activity of the truncated receptor. We found that the CB1-41 Gai1p1y2 complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells and that the protein complex is stable in .