Figure 4. Mutation of 5.38 confers G protein signaling bias in multiple GPCRs.

(A) The D3R-WT and D3R-F188^5.38A were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2-mVenus and GRK2. Dopamine-stimulated β-arrestin recruitment was assessed by BRET. (B) The D3R-WT and D3R-F188^5.38A were expressed in HEK293 cells with Goα1-Rluc8, β1, and γ2-mVenus. The cells were stimulated with dopamine and assayed for G protein activation by BRET. (C) Dopamine-stimulated β-arrestin recruitment was assessed for the D4R-WT-Rluc-8 and D4R-Y192^5.38A-Rluc8 as described in (A). (D) Dopamine-stimulated Go activation was assessed for the D4R-WT and D4R-Y192^5.38A as described in (B). (E) The β2R-WT and β2R-Y199^5.38A were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2-mVenus. Epinephrine-stimulated β-arrestin recruitment was assessed by BRET. (F) The β2R-WT and β2R-Y199^5.38A were expressed in HEK293 cells with Gα_s-RLuc8, β1, and γ2-mVenus. The cells were stimulated with epinephrine and assayed for G protein activation by BRET. (G) V2R-WT or the indicated V2R mutant were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2-mVenus and assayed for AVP-stimulated β-arrestin recruitment by BRET. (H) HEK293 cells expressing either V2R-WT or the indicated V2R mutant were assayed for AVP-stimulated cAMP accumulation using the TR-FRET-based Lance cAMP Detection kit. Data are expressed as a percentage of the maximum response for WT receptor. Average EC₅₀ and Emax values are found in table S6. All data points represent the mean ± SEM of 3–6 independent experiments performed in technical triplicate.