Stability and activity of a GPCR in vivo and in membrane mimetic environments.

摘要

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. The objective of this research was twofold: 1) understanding structural and environmental factors important for GPCR expression, trafficking and function, and 2) developing in vitro membrane-mimetic reconstitution environments to characterize ligand-binding kinetics and lipid-receptor interactions.;The role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the full-length human A2A adenosine receptor (A 2AR) was examined. To this end, systematic cysteine-to-alanine mutations were conducted to replace individual and paired cysteines in the first two extracellular loops. Although none of the disulfide bonds were essential (including the conserved disulfide bond) for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity.;There is growing evidence that ligand-binding rate constants could be better predictors of drug efficacy than affinity measurements. However ligand-binding kinetics are not well characterized, as they are more challenging to measure experimentally, and rarely investigated. Using fluorescence anisotropy we determined the association and dissociation rate constants for three unlabeled ligands (NECA, adenosine, and ZM 241385), in competition with fluorescent ligand FITC-APEC. We identified that the different affinities between two structurally similar agonists (NECA and adenosine) result from differences in their residence time (i.e. 1/koff). Furthermore, we determined that the higher affinity of ZM 241385 results primarily from a faster association rate.;Previous studies of A2AR purified using the detergent dodecylmaltoside (DDM), found that a cholesterol analog was critical for ligand binding and conformational stability of the receptor. Here, we show that A2AR purified using a short hydrocarbon-chain lipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine, DHPC) retains functionality and stability in the absence of added sterol. Additionally, longer chain lipids can be added to A2AR-DHPC to form bicelles, a biologically more relevant membrane-mimetic environment. Overall, the studies described in this thesis will be important as we begin to understand the emerging ligand-lipid-receptor-effector relationships.