Abstract
Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2, and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB2, here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB2 were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of 2H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.
Highlights
Heptahelical G protein-coupled receptors (GPCRs) are integral membrane proteins involved in a wide range of physiological processes including sensory transduction and cell-to-cell communication [1]
An expression of CB2 in E. coli as a fusion with maltose binding protein leads to production of a fully functional receptor located in the bacterial plasma membrane [5]
The data confirm that n-dodecyl-ß-D-maltopyranoside (DDM) in (1%) and 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS) (0.5%) with and without supplementation with 0.1% cholesteryl hemisuccinate (CHS) as used earlier [5,20] are most efficient in solubilizing CB2
Summary
Heptahelical G protein-coupled receptors (GPCRs) are integral membrane proteins involved in a wide range of physiological processes including sensory transduction and cell-to-cell communication [1]. GPCRs represent as much as 50% of pharmaceutical drug targets currently under development, the progress with structural studies has been relatively slow, in part due to the difficulties in obtaining large quantities of sufficiently pure, homogenous and functional protein. In addition to the availability of large quantities of purified receptor, structural methods require that the protein is sufficiently stable over extended periods of time. While solubilization in detergents is needed for extraction of GPCRs from cell membranes and chromatographic purification, the preservation of the structural integrity of receptors in micelles is a notoriously difficult task. The relatively few successful attempts to preserve the functional structure of purified GPCRs relied on a careful selection of mild solubilizing detergents as well as on supplementation of buffers used for purification with stabilizers such as lipids and ligands, adjustment of ionic strength and glycerol content. No general methodology for an efficient stabilization of GPCRs has been developed yet, and their low stability remains a major bottleneck for structural biology [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
