Abstract：
:The human cannabinoid receptor, CB1, has been difficult to purify in a functional form, hampering structural and biophysical studies. Here, we present our approaches for obtaining pure, detergent solubilized, functional CB1. We also discuss our site-directed fluorescence labeling (SDFL) methods for identifying different structural changes that CB1 can undergo upon binding different cannabinoid ligands. To identify optimal CB1 constructs for these studies (those with the best expression levels, solubility in detergent and function), we first screened various CB1-green fluorescent protein chimeras in a mammalian expression system. Once identified, we then tagged the best candidates with the 1D4 epitope (the C-terminus of rhodopsin) and purified them using a single-step immunoaffinity process. The resulting, highly pure proteins retain their ability to activate G-protein, and are ~85% functional, as assessed by radioligand binding studies. The SDFL studies involve introducing single cysteine residues at key places in the receptor, then labeling them with a small fluorophore, bimane. The spectral properties of the bimane probe are then monitored before and after addition of cannabinoid ligands. Changes in fluorescence of the attached probe indicate regions of the receptor undergoing conformational changes upon ligand binding. Together, these approaches set the stage for a deeper understanding of the structure and function of CB1. Access to pure, functional CB1 makes subsequent structural studies possible (such as crystallography and single-particle EM analysis), and the SDFL studies enable a better structural and mechanistic understanding of this key receptor and the dynamic changes it undergoes during activation and attenuation.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Fay JF,Farrens DLdoi
10.1016/bs.mie.2017.06.026subject
Has Abstractpub_date
2017-01-01 00:00:00pages
343-370eissn
0076-6879issn
1557-7988pii
S0076-6879(17)30183-0journal_volume
593pub_type
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