Facilitatory Interplay in α_1aand β₂Adrenoceptor Function Reveals a Non-G_qSignaling Mode: Implications for Diversification of Intracellular Signal Transduction

Abstract

Agonist occupied ␣ 1 -adrenoceptors (␣ 1 -ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress ␣ 1 -and ␤-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in ␣ 1A -and ␤ 2 -AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca 2ϩ mobilization to monitor in real time the activation of both G q and G s pathways. We found that coactivation of ␣ 1A -and ␤ 2 -AR by the nonselective agonist NE or via a combination of the highly selective ␣ 1A -AR agonist A61603 and the ␤-selective agonist isoproterenol led to increases in Ca 2ϩ influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca 2ϩ from intracellular stores. This effect became more evident upon examination of an ␣ 1A -AR variant exhibiting a partial defect in coupling to G q , and we attribute it to potentiation of a non G q -pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca 2ϩ entry channel blocker. We also found that stimulation with A61603 of a second ␣ 1A -AR variant entirely unable to signal induced no Ca 2ϩ unless ␤ 2 -AR was concomitantly activated. These results may be accounted for by the presence of ␣ 1A /␤ 2 -AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting ␣-and ␤-adrenoceptors.Given the diversity of physiological processes controlled by G protein-coupled receptors (GPCRs or 7-transmembrane receptors) and the large number of receptors from this family coexpressed in different tissues, it comes as no surprise that linear signal transduction models cannot accommodate the many observed pharmacological outcomes that follow receptor activation. It is generally understood that intracellular signal transduction pathways stemming from the activation of GPCRs interact significantly to add layers of complexity to their regulation, sometimes leading to novel signaling modes (Cordeaux and Hill, 2002). In addition, many receptors have been shown to couple to more than one G protein, and also initiate G protein-independent signaling upon stimulation (for review, see Gilchrist, 2007;Violin and Lefkowitz, 2007).Such GPCR signalin...

Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

“…2c). The measured EC 50 value for receptor internalization was ϳ0.8 M, which is comparable with the reported binding affinity between ISO and ␤ 2 ARs (Baker et al, 2003a;Hoffmann et al, 2004;Copik et al, 2009).…”

Targeting α- and β-Adrenergic Receptors Differentially Shifts Th1, Th2, and Inflammatory Cytokine Profiles in Immune Organs to Attenuate Adjuvant Arthritis

“…Whether this interaction involves multiple cell types and/or direct or indirect interplay between α- and β-ARs in the same cell types is unclear. Provocative new data (38, 39) indicate that β- and α-AR subtypes can form heterodimers that affect downstream signaling to regulate the inflammatory response. Similar research is warranted in our model to better understand β- and α-AR-mediated mechanisms regulating soft tissue swelling.…”

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

“…After stimulation by their ligands, α 1 -ARs activate intracellular effectors, including phospholipase C β (PLCβ), inositol trisphosphate, protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and calcium signals, often through a heterotrimeric G protein-dependent manner [14], [15]. An α 1A -AR variant, which was unable to couple to G q , could also induce calcium influx when coactivated by β 2 -AR [16]. Thus, α 1A -AR, even though uncoupled from G q , may remain competent for induction of signaling events through yet unknown pathways.…”