The cholecystokinin type 1 receptor (CCK1R) is a class A GPCR which is activated by the endogenous peptide ligand cholecystokinin (CCK). CCK1R has a wide range of roles in metabolism; including gastric motility, gallbladder contraction, and satiety. For these roles in metabolism the CCK1R is an attractive candidate for treating metabolic disorders such as obesity. Interestingly, CCK1R has been observed to be sensitive to membrane cholesterol content while the other CCK subtype, CCK2R, is not. Understanding CCK1R cholesterol sensitivity will not only be important for treating obesity, in which cholesterol levels can be irregular, but also may be applicable to other GPCRs.
Currently there are no atomic level resolution structures of CCK1R or CCK2R. In this study we aim to determine the inactive structure of CCK1R and the active structure in complex with heterotrimer G protein. Towards the active structure we have been able to express robust quantities of CCK1R and have optimised co-expression with heterotrimeric G protein (GS). Using the dominant negative GαS mutant (DNGs), we have been able to purify a complex with CCK/CCK1R/DNGs/NB35. The complex has been confirmed by negative stain TEM and will be used for structure determination by Cryo-EM.
An activate structure of CCK1R in complex with heterotrimeric G protein will help understand the mechanisms of CCK1R activation. Further, the structure will set the groundwork for exploring the cholesterol sensitivity of CCK1R, with use of CCK1R/CCK2R chimeric mutations and modification of cholesterol content.