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G protein-coupled receptors (GPCRs) represent the largest human receptor family with approximately 800 individuals and serve as a binding target for about 30% of all marketed drugs. They bind to various extracellular ligands such as neurotransmitters or hormones, to transduce signals into the cell without the need for passing the cell membrane. Depending on which of the four heterotrimeric G protein families these receptors preferentially couple to, specific physiological responses will be induced in a cell. Therefore, understanding the mechanism of selective G protein recognition and activation by receptors is of central importance.
Recently published structures of GPCR-G protein complexes have significantly enhanced the understanding of how GPCRs interact with G proteins. However, they are only snapshots which miss the complete temporal sequence of the multi-step coupling process. Moreover, they failed to reveal specific interaction sites between GPCRs and distinct Gα-subfamilies.
Therefore, in the first part of this thesis, the influence of subtype-specific Gα-structures on the stability of GPCR-G protein complexes and the subsequent activation by two Gq-coupled receptors was directly compared. For that reason, FRET-assays were performed in living cells to distinguish multiple Go and Gq-based Gα-chimeras in their ability to be selectively bound and activated by muscarinic M3 (M3R) and histaminic H1 receptors (H1R). Firstly, binding stabilities of Gα-chimeras were determined in permeabilized cells by comparing their dissociation kinetics from the Gq-coupled M3R and H1R under nucleotide-depleted conditions. Moreover, the activation potencies of Gα-chimeras were examined in intact cells by evaluating concentration-response curves that ultimately reflect physiologically relevant coupling efficiencies. For both receptors, we obtained results showing that binding and activation characteristics of crucial Gα-structures like the N-terminus including the αN/β1-hinge, the β2/β3-loop and the α5-helix can be transferred from one G protein class onto another. Furthermore, key selectivity determinants differ in their impact on selective binding to GPCRs and subsequent activation depending on the specific receptor.
In the second part of this thesis, structural elements in the receptor were investigated with regard to their influence on coupling-selectivity. Initial attempts to alter the M3R coupling-properties towards Gq or Go proteins by point mutations were without success. Therefore, the big third intracellular loop (ICL3) of the M3R was deleted which again did not influence the binding stabilities of Gq and Go proteins. However, swapping the ICL3 with the closely related Gi/o-coupled M2R (M3ICL3M2) revealed an enhanced binding to Gαo. Finally, the role of helix 8 for the generation of G protein coupling-preferences was investigated by creating chimeras between the M3R and M2R. Surprisingly, the M3R-based M3H8M2-chimera could not bind to Gαo anymore even though the M2R, containing the same helix 8, couples to Gi/o proteins readily. In line with this result, the stability of Gαo at the M2H8M3-chimera was distinctly enhanced and even Gq-binding was enabled.
Altogether, these findings provide new insights into the molecular basis of coupling-selectivity between various GPCRs and G proteins.