Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 24, Pages 17167-17178Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.464065
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Funding
- American Heart Association National Scientist Development Grant [13SDG14270009]
- McKay award
- Rackham Merit fellowship
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Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a G alpha subunit. We use these sensors to elucidate the influence of the highly conserved (E/D) RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D) RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D) RY motif ( ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the beta 2-AR (E/D) RY ionic lock enhances interaction with G(s). However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D) RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of beta 2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D) RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with G(s) and promotes G(i) binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.
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