Figure 3-. GPCR biology investigated through the use of ER/K linkers.

A) Detailed schematic design of GPCR-Gα α5-helix peptide (Gα peptide) sensors (left). Cartoon rendering of GPCR-Gα peptide sensor interactions (right).

B) Metoprolol stimulation of the indicated GPCR-Gα peptide sensors. Metoprolol (Meto), an inverse agonist of β1-AR signaling in the heart, suppresses the β2-AR-Gαs peptide interaction while stimulating interaction between β2-AR and Gαi peptide. In contrast, isoproterenol (Iso), a full agonist is shown to stimulate only the canonical β2-AR-Gαs peptide interaction.

C) Sequence alignment of Gαs and Gαq α5-helix, from which the Gα peptides are derived.

D,E) The indicated residues in panel C have been mutated to their counterpart residue from the other peptide. β2AR, is a Gαs coupled receptor (D) and shows diminished interaction with S-peptide mutated to look like Q-peptide. V1AR, a Gαq coupled receptor (E) shows enhanced interaction to S-peptide mutated to look like Q-peptide.

F) Cartoon rendering of GPCR-Gα protein sensors with different length linkers. These protein sensors have the full-length G protein instead of the Gα α5-helix alone.

G,H) β2-AR sensors tethered to either full length Gαs (G) or full length Gαq (H) with varying length ER/K linkers. Both sensors show decreases in cAMP production with increasing length of ER/K linkers.

For all experiments: N ≥ 3, *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001; n.s. not significant. Significance determined using Student’s t-test or ANOVA with Tukey’s post-hoc test where appropriate.

Panels A (left) and B adapted from Malik et al., JBC 2013. This research was originally published in the Journal of Biological Chemistry. Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells. J Biol Chem. 2013; 288(24):17167–78. © the American Society for Biochemistry and Molecular Biology.

Panels A(right), and C-E adapted from Semack et al., JBC 2016. This research was originally published in the Journal of Biological Chemistry. Structural elements in the Gαs and Gαq C-termini that mediate selective GPCR signaling. J Biol Chem. 2016; 19;291(34):17929–40. © the American Society for Biochemistry and Molecular Biology

Panels F-H adapted from Gupte et al., Priming GPCR signaling through the synergistic effect of two G proteins. PNAS 2017. 114(14):3756–3761.