Selectivity and efficiency of receptor mediated G protein activation
G protein coupled receptors (GPCR) regulate many important physiological functions by converting extracellular stimuli into the variety of biological responses. The spectrum of induced signalling pathways depends on the type of heterotrimetric G protein coupling to the receptor. A number of recent s...
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|Summary:||G protein coupled receptors (GPCR) regulate many important physiological functions by converting extracellular stimuli into the variety of biological responses. The spectrum of induced signalling pathways depends on the type of heterotrimetric G protein coupling to the receptor. A number of recent structural and computational studies could visualize and define motifs important for G protein-GPCR complex formation. However, the essential factor for the receptor-G protein selectivity has not been fully elucidated. In this research project it was hypothesized that G protein-receptor selectivity can be determined by G protein affinity, which can be numerically assessed as ternary complex stability. Interaction of G protein with the receptor can be subdivided into three major phases: G protein binding to the ligand-activated receptor, nucleotides exchange (GDP to GTP) on the Gα subunit which leads to ternary complex formation (ligand-receptor-G protein), and finally activation of the heterotrimeric G protein resulting in rearrangement of Gα and Gβγ subunits. Taking into account that ternary complex is stable only in the absence of nucleotides, I have developed a broadly applicable FRET-based assay to study receptor-G protein interaction in permeabilized transiently transfected cells under nucleotide depleted conditions. This approach allowed to quantitatively as both association and dissociation rates of G proteins from agonist-activated GPCRs. Activated by similar agonists, members of muscarinic acetylcholine receptor family (mAChRs) have been known to couple to different G protein classes. Therefore, mAChRs were taken as a model to study GPCR-G protein selectivity. G protein affinity was calculated as dissociation kinetics in response to withdrawal of agonist. Remarkably, the dissociation rate was the slowest for those complexes that contained G protein subtypes that are known to be most efficiently activated by the respective receptor. Specifically, dissociation of Gq protein from M3- and M1-Rs was significantly slower in comparison to Go, Importantly, the shift in concentration-response curves in steady-state experiments correlated well with the calculated Gq and Go affinities. Moreover, G protein activation measurements revealed quantitative correlation between coupling efficiency and affinity of Go and Gq to M3-R. This suggests that the stability of the receptor-G protein complex is an inherent property of both interaction partners and closely correlates with the ability of the receptor to activate the corresponding G protein subtype. In addition, by calculating the constants of association and dissociation, it has been shown that the affinity of the G protein to the receptor, but not the rate of ternary complex formation, determines the degree of GPCR-G protein selectivity.
Apart from mAChRs, this principle was also applicable for other GPCRs: µ-OR, β1-, β2-, and α2A-adrenegic receptors, TP-R and ETRB. Interestingly, each receptor revealed an individual pattern of G protein affinities. It was also observed that receptors known for high basal activity form extremely stable complexes with their regular interacting G proteins. The characterization of G protein affinities spectrum for such GPCRs was challenging. In this case, in order to quantify the ternary complex stability, the dissociation rate was modulated by the addition of constant low GTP concentrations. Thus, it was possible to show the difference between the affinities of individual members of G protein families. Moreover, the developed nucleotide-free method was used to test a range of orthosteric and allosteric mAChRs agonists with different affinities and efficiencies in terms of ternary complex stability. It was shown that the efficiency, but not the affinity, of the ligand to the receptor can be estimated as the rate of GPCR-G protein complex association. Calculated constant of association correlated with the efficiency of G protein activation and reflected the probability of the receptor to be stabilized in the active conformation by the particular agonist. Interestingly, the results of FRET experiments on permeabilized cells correlated with the data derived from FRET measurement on membranes. The last technique can be further used as a cell-free screening of different pharmacological agents effecting GPCR-G protein dynamics. To conclude, my findings shed new light on the mechanisms which determine the receptor-G protein selectivity.|
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