Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G-protein fusion protein.
We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies of the human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic gene containing unique restriction sites spaced roughly 50-100 bases apart to facilitate rapid mutagenesis and cloning. A nine amino acid epitope tag (from the rhodopsin C-terminus) is also present in the shCB1 C-terminal tail to enable detection and purification using the monoclonal antibody 1D4. We find that that the shCB1 gene can be transiently expressed in COS cells with yield of approximately 10-15 micro g receptor per 15 cm plate and is wild type like in its ability to bind cannabinoid ligands. Our confocal microscopy studies indicate shCB1 targets to the membrane of HEK293 cells and is internalized in response to agonist. To facilitate functional studies, we also made a chimera in which the C-terminus of shCB1 was fused with the N-terminus of a G-protein alpha subunit, Galphai. The shCB1/Galphai chimera shows agonist stimulated GTPgammaS binding, and thus provides a simplified way to measure agonist induced CB1 activation. Taken together, the shCB1 and shCB1/Galphai gene constructs provide useful tools for biochemical and biophysical examinations of CB1 structure, activation and attenuation.
Farrens
D L
DL
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239-3098, USA.
Dunham
T D
TD
Fay
J F
JF
Dews
I C
IC
Caldwell
J
J
Nauert
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon; D000939:Epitopes / Q000032:analysis / Q000235:genetics; D019204:GTP-Binding Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010880:Piperidines / Q000378:metabolism; D011720:Pyrazoles / Q000378:metabolism; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D000077285:Rimonabant; D014162:Transfection
D000906:Antibodies; D003062:Codon; D000939:Epitopes; D008024:Ligands; D010880:Piperidines; D011720:Pyrazoles; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins; D000077285:Rimonabant