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C‐terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity
Author(s) -
Chillakuri Chandramouli Reddy,
Reinhart Christoph,
Michel Hartmut
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.06132.x
Subject(s) - sf9 , g protein , g protein coupled receptor , cannabinoid , receptor , cannabinoid receptor , 5 ht5a receptor , enzyme linked receptor , heterotrimeric g protein , microbiology and biotechnology , biology , chemistry , biochemistry , spodoptera , antagonist , gene , recombinant dna
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 Gα i1 β 1 γ 2 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 Gα i1 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/Gα i1 β 1 γ 2 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.