PubAg

Main content area

C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

Author:
Chillakuri, Chandramouli Reddy, Reinhart, Christoph, Michel, Hartmut
Source:
FEBS journal 2007 v.274 no.23 pp. 6106-6115
ISSN:
1742-464X
Subject:
agonists, antagonists, cannabinoids, cell membranes, dimerization, energy transfer, fluorescence, insects, proteins, solubilization, yields
Abstract:
We have investigated the existence of a precoupled form of the distal C-terminal 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 Gαi₁β¹γ² proteins were colocalized in the cells. GTPγS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Gαi₁ protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Gαi₁β¹γ² 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 detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.
Agid:
3046299