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, beta(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, beta-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.
Highlights
The 2-adrenergic receptor (2AR)2 and prostanoid receptors (EP2R and EP4R) are Gs-linked receptors that, upon agonist occupancy, activate adenylyl cyclases (ACs) to produce a
To assess the specific role of PDE4 in shaping sub-plasmalemmal cAMP dynamics and in down-regulating signaling efficiency during 2AR activation, cells were treated with an adenoviral vector expressing a mutant ␣-subunit of the rat olfactory cyclic nucleotide-gated channels (CNGCs) (C460W/E583M CNGC)
Activity of the CNGCs in individual HEK293 cells was assessed by monitoring Ca2ϩ influx as a function of cAMP-mediated channel activation
Summary
The 2-adrenergic receptor (2AR) and prostanoid receptors (EP2R and EP4R) are Gs-linked receptors that, upon agonist occupancy, activate adenylyl cyclases (ACs) to produce a. Differential agonist actions are 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 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). In complementary experiments we overexpressed catalytically inactive, “dominant negative” versions of PDE4D isoforms, to assess the role of these proteins during both isoproterenol- and prostaglandin-evoked receptor activation By displacing such cognate, anchored endogenous PDE4 isoforms from their functionally relevant sites, these constructs provide a unique insight into signaling functions of spatially constrained PDE4 isoforms [14, 15]
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