A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging.

Yao Chen,Gary Yellen,Jessica L Saulnier,Bernardo L Sabatini

doi:10.3389/fphar.2014.00056

Abstract

Neuromodulators have profound effects on behavior, but the dynamics of their intracellular effectors has remained unclear. Most neuromodulators exert their function via G-protein-coupled receptors (GPCRs). One major challenge for understanding neuromodulator action is the lack of dynamic readouts of the biochemical signals produced by GPCR activation. The adenylate cyclase/cyclic AMP/protein kinase A (PKA) module is a central component of such biochemical signaling. This module is regulated by several behaviorally important neuromodulator receptors. Furthermore, PKA activity is necessary for the induction of many forms of synaptic plasticity as well as for the formation of long-term memory. In order to monitor PKA activity in brain tissue, we have developed a 2-photon fluorescence lifetime imaging microscopy (2pFLIM) compatible PKA sensor termed FLIM-AKAR, which is based on the ratiometric FRET sensor AKAR3. FLIM-AKAR shows a large dynamic range and little pH sensitivity. In addition, it is a rapidly diffusible cytoplasmic protein that specifically reports net PKA activity in situ. FLIM-AKAR expresses robustly in various brain regions with multiple transfection methods, can be targeted to genetically identified cell types, and responds to activation of both endogenous GPCRs and spatial-temporally specific delivery of glutamate. Initial experiments reveal differential regulation of PKA activity across subcellular compartments in response to neuromodulator inputs. Therefore, the reporter FLIM-AKAR, coupled with 2pFLIM, enables the study of PKA activity in response to neuromodulator inputs in genetically identified neurons in the brain, and sheds light on the intracellular dynamics of endogenous GPCR activation.

Highlights

Neuromodulators such as dopamine, serotonin and opioids have profound effects on neurons, circuits and behavior (Hikosaka et al, 2008; Kreitzer and Malenka, 2008; Le Merrer et al, 2009)
GENERATION AND COMPARISON OF CONSTRUCTS FOR 2-photon fluorescence lifetime imaging microscopy (2pFLIM) IMAGING OF NET protein kinase A (PKA) ACTIVITY AKAR3 and its derivatives were successfully used for ratiometric imaging of PKA activity (Allen and Zhang, 2006; Vincent et al, 2008; Depry et al, 2011; Lam et al, 2012), they are not suitable for imaging in brain tissue with 2pFLIM due to spectral bleedthrough and the properties of the donor fluorophore
In the presence of the PKA inhibitor peptide PKI (Ashby and Walsh, 1972, 1973; Dalton and Dewey, 2006), Fluorescence Lifetime Imaging Microscopy (FLIM)-AKAR did not respond to addition of forskolin or H89 (Figures 5D–F). These results demonstrate that FLIM-AKAR does not respond to PKC, and is specific for PKA following adenylate cyclase (AC) activation by forskolin

Summary

Introduction

Neuromodulators such as dopamine, serotonin and opioids have profound effects on neurons, circuits and behavior (Hikosaka et al, 2008; Kreitzer and Malenka, 2008; Le Merrer et al, 2009). Extensive biochemical characterization has identified protein kinase A (PKA) as a convergent site of action for many neuromodulators and neurotransmitters (Greengard, 2001). Most neuromodulators exert their function via G-protein-coupled receptor (GPCRs); neurotransmitters including glutamate and GABA can act via metabotropic receptors that are GPCRs. GPCRs coupled to Gαs and Gαi produce up- and down-regulation of adenylate cyclase (AC) activity, respectively. Gαs- and Gαicoupled GPCRs bidirectionally change PKA activity (Greengard, 2001). PKA can act as a potential integrator of diverse cellular inputs to mediate synaptic and cellular changes

Methods

Results

Conclusion