Characterization Of The Alpha-Kap Fret Biosensor To Determine Compartmentalized Beta-Adrenergic Receptor Camp Signaling In Distinct Intracellular Locations

The sympathetic nervous system helps maintain cardiac homeostasis primarily through the activation of β1 and β2-adrenergic receptors (β-ARs). Both types of receptors rely on the production of the diffusible second messenger 3',5'-cyclic adenosine monophosphate (cAMP) to mediate a variety of cellular functions. However, both receptors do not produce the same cAMP-dependent responses. One explanation for these differences is that cAMP is compartmentalized, but what is responsible for this compartmentation is not well understood. FRET-based biosensors targeted to different subcellular locations were used to test the hypothesis that variation in the distribution of β1- and β2-ARs in the plasma membrane contribute to differences in the subcellular pattern of cAMP production. The novel a-Kinase Anchoring Protein (EPAC2-α-KAP) FRET biosensor was created and characterized to determine cAMP production in proximity to the free sarcoplasmic reticulum, an intracellular site hypothetically devoid of β2-AR cAMP production. In combination with other targeted FRET biosensors, distinct cAMP signals were observed in response to selective β1- and β2-ARs stimulation. All the FRET probes demonstrated cAMP production in response to β1-AR specific stimulation but the α-KAP probe did not respond to β2-AR specific stimulation as predicted. The results of this study should provide a better understanding of the differences in β1- and β2-AR regulation of cAMP signaling and the importance these differences plays in sympathetic functional responses.