A complex interplay of various intracellular motifs determines G protein binding and activation of muscarinic receptors.

G protein coupled receptors (GPCRs) mediate intracellular signaling by selectively activating heterotrimeric G proteins. While certain GPCRs exhibit a high specificity toward particular G protein subtypes, other GPCRs display promiscuous signaling by engaging interaction with multiple G protein families. Molecular determinants underlying the selectivity or promiscuity of the receptors remain incompletely understood. In the present study, we investigate various structural motifs within the intracellular domains of Muscarinic receptors to assess their role in both Gα subunit binding and activation. To this end, we generated chimeric receptors and applied both FRET- and BRET-based assays to monitor G protein binding and activation. Our study demonstrates that the determination of G protein coupling selectivity is not defined by single motifs or amino acids but rather by a complex interplay of various intracellular motifs affecting binding or/and subsequent activation. These results provide new insights into the structural basis of GPCR-G protein specificity.