Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β-adrenergic receptor (βAR) and the cholecystokinin (CCK) receptor subfamily. The βAR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous βAR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCKR shows functional sensitivity to membrane cholesterol while CCKR does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with βAR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-T) shows that mutation of a conserved CRAC sequence on TM7 of the βAR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.