Temporal Modulation of a GPCR Pathway Elucidates Band-Pass Processing for the Downstream Signaling and Transcription Factor Activation

Abstract

G-protein coupled receptors (GPCRs) are major pharmaceutical targets. Quantitative understanding of GPCR signaling pathway architecture has been limited by the temporal observability space available in the dose-response studies. We use a microfluidic platform that delivers temporally modulated step and pulsatile ligands to single HEK293 cells with simultaneous real-time assessment of downstream calcium signal and calcium-regulated transcription factor activation. We pair these experiments with computational modeling to delineate the mechanism of frequency and amplitude modulation by GPCRs. We find that the pulsatile ligand input, when timed appropriately, leads to better recovery of the calcium signal as well as greater downstream transcription factor activation as compared to step changes in the ligand concentration. Further, we also show that receptor kinetics and the dynamics of downstream signaling combine to form a band-pass regime of frequencies for which the signaling is significantly enhanced. Computational modeling suggests that the band-pass nature of signaling pathways may be a generic theme wherein an upstream module acts as a low-pass filter to distinguish signal from noise, and the downstream module acts a high pass filter to amplify signals received. We further delineate the low pass filter characteristics of the GPCR in single cells and show that cell-to-cell variability may lead to distinguishable downstream responses that may not be obvious by looking at the population scale response. These findings may facilitate the design of therapeutic interventions and the development of enhanced in vitro cell culture protocols.