Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

Abstract

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G s -coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G s -coupled receptor pathways, ␤ 2 -adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, ␤-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G s -linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.The ␤ 2 -adrenergic receptor (␤ 2 AR) 2 and prostanoid receptors (EP2R and EP4R) are G s -linked receptors that, upon agonist occupancy, activate adenylyl cyclases (ACs) to produce a diverse number of cAMP-dependent biological actions. Many tissues types, including airway epithelium and cardiac muscle, endogenously express both classes of G-protein-coupled receptor to generate cellular increases in cAMP following appropriate stimuli (1, 2). Differential agonist actions are thus conjectured to be permitted via compartmentalization of cAMP signals to regions close to individual G-protein-coupled receptor subtypes (3, 4). These local cAMP signals can then mediate specific downstream events through the regulation of neighboring effector molecules such as protein kinase A (PKA), exchange protein directly activated by cAMP, and cyclic nucleotide-gated channels (CNGCs). Temporally distinct, localized cAMP signals can be produced by two main factors: firstly, rapid cAMP hydrolysis by spatially organized cAMP phosphodiesterases (PDEs) (3-9) and, secondly, rapid receptor uncoupling or "desensitization" causing reduced G s -coupled receptor responsiveness to subsequent or prolonged agonist application (10, 11).The study of separate pools of cAMP deriving from different receptors in the same cell is most directly examined at the single cell level. Recent real-time, single cell approaches have provided some insight into the composition of signaling complexes whose tethered PDEs serve to shape a localized cAMP signal (9, 12, 13). However, the characteristics of the other key regulators comprising rapid attenuation of cAMP signal and receptor desensitization have not been addressed a...

Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

“…AC2 is also stimulated in a PKC-independent manner by Ga s in response to activation of G s -coupled receptors and directly via the small molecule AC activator forskolin. PGE 2 is known to bind and activate the G s -coupled EP2/4 prostanoid receptors that are endogenously expressed in HEK293 cells (Willoughby et al, 2007;Bogard et al, 2012). As expected, PGE 2 treatment resulted in a concentration-dependent increase in cAMP accumulation in HEK-hAC2 cells (EC 50 : 160 6 78 nM, n 5 3).…”

Mechanism regulating proasthmatic effects of prolonged homologous β2-adrenergic receptor desensitization in airway smooth muscle

“…Interestingly, in this context, it has been reported that PKA activation can acutely reverse the reported inhibitory phosphorylation of PDE4D5 by ERK1/2 (15). Moreover, recent studies have demonstrated that the acute cAMP responses detected in subcellular microdomains of HEK-293 cells exposed to a ␤2AR or prostanoid receptor agonist are rapidly attenuated by PKA-dependent PDE4 activity (notably involving PDE4D5), which mediates homologous desensitization of these receptors to subsequent agonist administration (36,37). While potentially also important in ASM under conditions associated with acute homologous ␤2AR desensitization, it is unlikely that the latter posttranslational mechanisms involving acute PKA-dependent activation of PDE4 activity significantly contributed to the upregulated PDE4 activity detected herein following prolonged ␤2AR agonist exposure, given that the enhanced PDE4 activity was completely ablated by inhibiting either de novo mRNA or protein synthesis with actinomycin D or cycloheximide, respectively (Fig.…”

Prolonged heterologous β2-adrenoceptor desensitization promotes proasthmatic airway smooth muscle function via PKA/ERK1/2-mediated phosphodiesterase-4 induction

“…In relation to these observations, it is noteworthy that, apart from upregulating PDE4D5 transcription, PKA is also known to directly stimulate PDE4D5 activity, as well as that of other long PDE4D isoforms, by phosphorylating a regulatory site within the amino-terminal region of these isoforms (9,24). Moreover, in this context, recent studies have demonstrated that the acute cAMP responses detected in subcellular microdomains of HEK293 cells exposed to a ␤2AR or prostanoid receptor (EP2 and EP4) agonist are rapidly attenuated by PKA-dependent PDE4 activity (notably involving PDE4D5), which also mediates acute homologous desensitization of these receptors to subsequent agonist administration, as well as acute heterologous desensitization of the ␤2AR following exposure to an EP2/EP4 agonist (48,49). Of significance, this rapid PKA-dependent/PDE4D-mediated attenuation of cAMP signaling and desensitization to ␤2AR and EP2/EP4 agonists was not detected in the presence of receptor-independent activation of adenylyl cyclase in forskolin-exposed HEK293 cells (38).…”