Purification of Functional CB1 and Analysis by Site-Directed Fluorescence Labeling Methods

Jonathan F. Fay, David Farrens

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages343-370
Number of pages28
Volume593
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume593
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Allosteric ligands
  • Biased signaling
  • Cannabinoid receptor purification
  • CB
  • GPCR dynamics and conformational changes
  • GPCR purification
  • GPCR structural studies
  • SDFL bimane fluorescence

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Fay, J. F., & Farrens, D. (2017). Purification of Functional CB1 and Analysis by Site-Directed Fluorescence Labeling Methods. In Methods in Enzymology (Vol. 593, pp. 343-370). (Methods in Enzymology; Vol. 593). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2017.06.026