The Beta 1 and Beta 2 Adrenergic Receptors Exhibit Differential Coupling to the Cyclic AMP Sensor NCS‐Rapgef2

The beta 1 and beta 2 adrenoceptors (β1 and β2 AR) are GPCRs for the catecholamines norepinephrine and epinephrine. Both of these receptors couple to Gsα. Their activation causes cAMP elevation, which in turn controls cellular signaling through its downstream effectors PKA and the cAMP guanine nucleotide exchange factors (GEFs) Epac1 and Epac2. The 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 recently characterized a neuronal and endocrine‐specific cAMP sensor, a GEF related to Epac 1 and 2, and to the previously characterized non‐cAMP‐activated GEF PDZ‐GEF1, which we have named NCS (neuritogenic cyclic AMP sensor)‐Rapgef2 (a protein product of the Rapgef2 gene). This sensor mediates activation of ERK leading to neuritogenesis in the PC12 and NS‐1 neuroendocrine cell lines (Emery et al., Sci. Signal. 6, ra51, 2013; Emery et al., J. Biol. Chem. 289: 10125, 2014). We have created NS‐1 cell lines stably expressing either β1AR or β2AR, and examined signaling to each of the three cAMP sensors present in these cells following treatment with isoproterenol. In β1AR‐expressing cells, agonist treatment caused activation of all measurable cAMP‐dependent signaling pathways in these cells: Epac2/p38‐dependent growth arrest; PKA‐dependent CREB phosphorylation; and NCS‐Rapgef2/ERK‐dependent neuritogenesis. In contrast, agonist stimulation of β2AR‐expressing cells caused isoproterenol‐initiated Epac2/p38‐dependent growth arrest and PKA‐dependent CREB phosphorylation, but did not couple to NCS‐Rapgef2/ERK‐dependent neuritogenesis. To compare the desensitization profiles of the two receptors, a biosensor that allowed for continuous real‐time cyclic AMP measurements was co‐expressed in each cell line (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. Unlike the mode of ERK phosphorylation observed following β1AR activation (NCS‐Rapgef2‐dependent), ERK activation elicited by β2AR (NCS‐Rapgef2‐independent) most likely occurs in a cellular compartment restricted from transcriptional regulation, which is required for neuritogenesis in this cell type (Ravni et al., Mol. Pharmacol. 73: 1688, 2008). We conclude that there is an inverse relationship between adrenergic receptor desensitization, and engagement of NCS‐Rapgef2 of sufficient duration to support the sustained activation of ERK necessary to promote neuritogenesis in NS‐1 cells. We notice the same inverse relationship in receptors for dopamine (D1) and adenosine (A2A), as well as the neuropeptides PACAP, VIP, and GLP‐1 (PAC1, VPAC1, VPAC2, and GLP‐1R).Support or Funding InformationThis work was supported by NIMH Intramural Research Program Project ZIAMH002386 and by a 2014 NARSAD Young Investigator Grant to A.C.E. from the Brain and Behavior Research Foundation (Grant 21356).