Designing a Constitutively Active G Protein-Coupled Receptor Mutant from an Inactive One and Vice Versa

Abstract

Cells convert a diversity of extracellular signals (like hormones) into physiological responses by activating G protein-coupled receptors (GPCRs) to trigger mainly G protein- and/or β-arrestin-mediated signaling pathways. This pleiotropic activity of GPCRs is enabled by their multiple distinct conformations each of which may be attributable to a specific functional state of the receptor. Many GPCRs exhibit measurable levels of basal activity in the absence of any extracellular stimuli. As a first step to understand GPCR activation, this study focuses on the conformational changes that underlie the basal activity of the CB1 cannabinoid GPCR by designing mutants that activate an inactive receptor or inactivate an active receptor. The modulation of this "basal conformational functional landscape" by extracellular stimuli can then provide a more complete picture of GPCR activation. The GTPγS assay data have shown that mutation of the threonine residue to alanine at position 210 of the constitutively active CB1 receptor makes the wild-type (WT) receptor inactive, whereas the T2101 or the L207A mutant displays high constitutive activity. We used a de novo GPCR structure prediction methodology to predict the structures of the WT receptor and its three mutants (T21OA, T21OI and L207A). The resulting structures, that are consistent with known conformational changes during GPCR activation, showed that a novel ionic lock (salt bridge interaction) between transmembrane (TM) helices 2 and 6 keeps the T210A mutant inactive. This observation was then used to design two active double-mutant receptors lacking the TM2+TM6 ionic lock, based on the inactive T21OA mutant. The GTP-γS assay data showed that these double mutants gained constitutive activity. The active L207A mutant that lacked the TM2+TM6 ionic lock was then used to design an inactive double-mutant receptor that had that ionic lock. The inactivity of this double mutant was also confirmed by the GTPγS assays. Designing active mutant forms from the inactive receptor and vice versa has provided a much clearer understanding of the critical conformational changes that occur upon the basal activation of the CB1 receptor. This has important relevance for the activation of other GPCRs and also sets the stage to understand how various ligands (from inverse agonists to agonists) can modulate this conformational landscape to disrupt or enhance receptor activation.

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