Persistence of Cyclic AMP Signaling Determines Gs‐coupled GPCR linkage to ERK activation
Authors: Emery Andrew Clayton , Eiden Maribeth V. , Xu Wenqin , Eiden Lee E. 
Published: June 2021

The beta 1 and beta 2 adrenoceptors (β1 and β2 AR) are GPCRs for the catecholamines norepinephrine and epinephrine. These receptors couple to Gs to cause cAMP elevation. Cyclic AMP then mediates intracellular signaling by activating the cAMP sensors PKA and the cAMP‐GEFs (guanine nucleotide exchange factors) Epac1 and Epac2. β2AR has also been shown to signal via engagement of β‐arrestin, which acts as a scaffold for a variety of signaling proteins, including the MAP kinase ERK. We have characterized an additional GEF that is activated by cAMP, and its expression is restricted to neuronal and endocrine cell types and tissues (Jiang et al., eNeuro, ENEURO.0248–17.2017). We have termed this signaling molecule NCS (neuritogenic cyclic AMP sensor)‐Rapgef2 (a protein product of the Rapgef2 gene). NCS‐Rapgef2 serves as the functional linkage between cAMP elevation and ERK phosphorylation, which leads to neuritogenesis in the neuroendocrine cell lines NS‐1 and PC12 (Emery et al., Sci. Signal. 6, ra51, 2013; Emery et al., J. Biol. Chem. 289: 10125, 2014).We generated NS‐1 cell lines that stably express either β1AR or β2AR to examine agonist‐induced signaling to each of the three cAMP sensors present in this cell type: PKA, Epac2 and NCS‐Rapgef2. In β1AR‐expressing cells, treatment with the agonist isoproterenol led to activation of all three of the major neuroendocrine cAMP‐dependent signaling pathways: Epac2/p38‐dependent growth arrest; PKA‐dependent CREB phosphorylation; and NCS‐Rapgef2 and ERK‐dependent neurite elongation. In contrast, agonist stimulation of β2AR‐expressing cells caused only Epac2/p38‐dependent growth arrest and PKA‐dependent CREB phosphorylation, but not result in NCS‐Rapgef2/ERK‐dependent neuritogenesis. To compare the desensitization profiles of the two receptors, cells were transduced with a cAMP biosensor (split luciferase PKA‐R) which enabled continuous real‐time measurements of cAMP abundance (Emery et al., Peptides, 79: 39, 2016). In β2AR‐expressing cells, the maximal effect of isoproterenol on cAMP was observed after 10 minutes of treatment and decreased rapidly thereafter. In contrast, isoproterenol‐dependent β1AR activation caused persistent cAMP elevation, observed at approximately maximal levels at least 40 minutes following agonist addition. The mode of ERK phosphorylation observed following β1AR activation (NCS‐Rapgef2‐dependent) results in gene transcription, including up‐regulation of Egr‐1, required for neuritogenesis (Ravni et al., Mol. Pharmacol. 73: 1688, 2008), while ERK activation elicited by β2AR (NCS‐Rapgef2‐independent) does not have this signaling property. That β1AR and β2AR stimulation by catecholamines both cause ERK activation, but presumably in two separate cellular compartments, may help to explain differential stimulation of physiological events elicited by these two receptors in a variety of cells and tissues.Support or Funding InformationThis work was supported by National Institute of Mental Health Intramural Research Program Grants 1ZIAMH002386 (to L.E.E.) and 1ZIAMH002592 (to M.V.E.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.