Fig. 1. Characterization of a G_s-biased PTH analog generated by amino acid isomerization.

(A and B) Concentration-response curves for PTH^WT, LA-PTH, and PTH^7d in cAMP accumulation (A) or in β-arr1 or β-arr2 recruitment (B) assays. Data are means ± SEM from N = 5 independent experiments.

(C) Time course and integrated response of β-arr2 recruitment measured by FRET in HEK293 cells transiently expressing PTHR^CFP and β-arr2^YFP following brief stimulation with 10 nM PTH^WT or PTH^7d. Data are means ± SEM from N = 3 independent experiments with n = 28–33 cells per experiment. ***, P < 0.005 by t-test.

(D) Recruitment of endogenous β-arr1 and β-arr2 (βarr1/2) to PTHR detected by coimmunoprecipitation with HA-tagged PTHR (^HAPTHR) stably expressed in HEK293 cells. Cells were challenged with 100 nM of either PTH^WT, LAPTH, or PTH^7d. Blot is representative of 2 independent experiments.