Conformational Landscape Governing the Constitutive Activity of GPCRs

Abstract

The pleiotropy of G protein-coupled receptors (GPCRs) is enabled by their conformational flexibility during activation. Many GPCRs exhibit constitutive activity, whose structural basis is not understood. This study explored the conformational changes that underlie the constitutive activity of the CB1 cannabinoid GPCR by designing constitutively active mutants starting from an inactive CB1 mutant and by designing inactive mutants starting from an active mutant. Ligand binding and GTPgS assay data (for G protein coupling) had suggested that T210A mutant of the CB1 receptor was inactive and two mutants T210I and L207A were more active than the wild-type (WT) receptor. Structure prediction of the WT and mutant forms resulted in conformational changes consistent with known changes during GPCR activation. It identified a unique salt-bridge interaction in the CB1 inactive mutant T210A between an Arg residue on transmembrane helix 2 (TM2) and a conserved Asp residue on TM helix 6 (TM6), which was proposed to keep the receptor fully inactive [Scott et al. (2013). Protein Science 22:101]. The WT and other active receptor mutants lacked this interaction. To test this hypothesis, this salt-bridge interaction was disrupted by designing two constitutively active double-mutants lacking the TM2+TM6 ionic lock, starting from the inactive T210A mutant. The GTPgS assay data confirmed that both these mutants were constitutively active. The active L207A mutant that lacked the TM2+TM6 ionic lock was then used to design an inactive double-mutant receptor that possessed that TM2+TM6 ionic lock. The inactivity of this double mutant was confirmed by GTPgS assays, and then reversed by adding a third mutation to rescue some of the constitutive activity [Ahn et al. (2013) Proteins 81:1304]. These data strongly support the role of TM2+TM6 salt-bridge interaction in keeping the receptor inactive and also show that the constitutive activity of this receptor is controlled by distinct changes in salt-bridge interactions in the TM region. These changes underlying constitutive activity provide a "conformational landscape" that can be modulated by extracellular stimuli like hormones to provide a more complete structural picture of GPCR activation.

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