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-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 Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(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/Galpha(i1)beta(1)gamma(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.
Chillakuri
Chandramouli Reddy
CR
Department of Molecular Membrane Biology, Max-Planck-Institute for Biophysics, Frankfurt/Main, Germany.
Reinhart
Christoph
C
Michel
Hartmut
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008970:Molecular Weight; D010880:Piperidines / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D011720:Pyrazoles / Q000494:pharmacology; D011869:Radioligand Assay; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D000077285:Rimonabant; D018411:Spodoptera / Q000166:cytology* / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000378:metabolism
D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D043562:Receptors, G-Protein-Coupled; D014760:Viral Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D000077285:Rimonabant