β2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway

Abstract

Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β₂AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα_s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca²⁺ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β₂AR activation leads to robust Ca²⁺ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP₃) receptors. This pathway did not involve cAMP, Gα_s, or Gα_i or the participation of the other members of the canonical β₂AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca²⁺ mobilization by β₂AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs.

Keywords: G-protein-coupled receptor (GPCR); adrenergic receptor; calcium intracellular release; cell signaling; cyclic AMP (cAMP); β2-adrenergic receptor.

Figure 1.

Endogenous β₂AR activation increased cytoplasmic [Ca²⁺] in HEK cells. Continuous changes in Fluo-4 fluorescence intensity with time (a and c) or peak increases in intensity as a function of drug concentration (b and d–h) are plotted. a, NE treatment increased cytoplasmic [Ca²⁺]. b, NE response is mimicked by AR agonists epinephrine (Epi) and isoproterenol (ISO) and the β₂AR-selective terbutaline (Ter). c, the β-AR inhibitor propranolol and the β₂AR-selective ICI 118,551 do not suppress P₂Y receptor signaling. d and e, β-adrenergic inhibitors suppress responses to ISO. f and g, α-adrenergic inhibitors do not suppress ISO responses. h and i, loss of Ca²⁺ response in cells lacking β₂AR and restoration by β₂AR expression. h, β₂AR deletion mutant cells (KO) were transfected with pcDNA3.1 or HA-tagged β₂AR and tested for Ca²⁺ responses over a range of ISO concentrations. i, β₂AR deletion mutant cells (KO) or wildtype (WT) cells were treated with 10 μm ISO at the indicated times, and Ca²⁺ responses were monitored over time. For all panels Ca²⁺ traces represent three or more independent experiments; error bars indicate internal replicate S.E. Dose responses are the averages of three or more independent experiments, and error bars indicate S.E. AU, absorbance units.

Figure 2.

The β₂AR induced calcium response is due to release from the endoplasmic reticulum. a and b, chelation of extracellular Ca²⁺ with EGTA does not eliminate β₂AR-mediated Ca²⁺ mobilization. c, depletion of endoplasmic reticulum Ca²⁺ by treatment with thapsigargin (TG) for 5 min nearly eliminates signaling with ISO. d, treatment with InsP₃R inhibitor 2-APB and PLC inhibitor U73122 for 1 h suppresses signaling with ISO. Ca²⁺ traces represent three or more independent experiments, and error bars indicate internal replicate S.E. AU, absorbance units.

Figure 3.

PKA did not mediate the β₂AR calcium response. a, 1 min of 8-Br-cAMP treatment did not mimic ISO response. b, 30 min of 8-Br-cAMP treatment did not mimic ISO response. c, 1 min of ISO significantly raised pCREB levels (**, p = 0.0065), whereas 1 min of 8-Br-cAMP treatment did not significantly raise pCREB levels (NS). d, 30 min of ISO (**, p = 0.0056) and 8-Br-cAMP treatment (*, p = 0.0133) significantly elevated intracellular pCREB. e, treatment with PKA inhibitors KT-5720 and H-89 for 1 h did not suppress signaling with ISO. Ca²⁺ traces represent three or more independent experiments, and error bars indicate internal replicate S.E. pCREB/CREB graphs are the averages of three independent experiments; error bars indicate S.E. Full blot images are shown in supplemental Fig. S1. AU, absorbance units.

Figure 4.

The β₂AR calcium response was not mediated by AC or cAMP. a, Gα_i activation did not inhibit ISO signals. b, Gα_i activation induced changes in membrane potential. c, treatment with PTX abolished μOR-induced membrane potential changes. d–g, treatment with AC inhibitors SQ 22,536 and ddAd did not suppress signaling with ISO, even at 1 mm, although AC inhibitor treatment significantly suppressed cAMP formation (* = p = 0.0298 and ** = p = 0.0059) (f). h and i, IBMX treatment did not potentiate signaling with ISO. Ca²⁺ traces represent three or more independent experiments, and error bars indicate internal replicate S.E. Dose responses and bar graphs are the averages of three or more independent experiments, and error bars indicate S.E. AU, absorbance units.

Figure 5.

β₂AR did not couple to Gα_s or Gα_i to initiate the calcium response. a, treatment with the Gα_s CTX did not potentiate signaling with ISO. b, treatment with CTX significantly increased cytoplasmic cAMP (***, p = 0.0004). c, treatment with the Gα_i PTX did not suppress signaling with ISO. d, treatment with PTX completely eliminated changes in membrane potential induced by the dopamine-2 receptor upon the addition of dopamine (DA). Ca²⁺ and membrane potential traces represent three or more independent experiments, and error bars indicate internal replicate S.E. The bar graphs are the averages of three or more independent experiments, and the error bars indicate S.E. AU, absorbance units.

Figure 6.

Supporting evidence against Gα_q coupling to β₂AR in TRPC4 β-expressing HEK-293 cells. Treatment with ISO did not mimic the membrane potential response of the Gα_q-coupled M₃ muscarinic receptors activated with carbachol. Ca²⁺ traces represent three or more independent experiments, and error bars indicate internal replicate S.E. Fig. 4 demonstrates robust membrane potential changes in response to μOR stimulation with DAMGO under these conditions without PTX treatment and robust Ca²⁺ release in response to isoproterenol stimulation under these conditions in these cells. AU, absorbance units.