Here we have used transcriptome analysis of primary neonatal

Here, we have used transcriptome analysis of primary neonatal rat cardiomyocytes treated with either the ETR agonist endothelin-1 or the α1-AR agonist phenylephrine to assess differences in their respective signalling networks, and further probed these differences using a panel of fluorescent resonance energy transfer (FRET)- and Hexamethonium Bromide sale resonance energy transfer (BRET)-based biosensors. We also used genetically-encoded biosensors targeted to specific cellular compartments to compare differential signalling by distinct GPCR populations. These experiments revealed unexpected specificity in signalling function, both among the receptor subtypes tested and between subcellular compartments.
Methods
Results
Discussion Here we have shown that two GPCR subtypes thought to trigger similar signalling events by coupling to Gαq in fact regulate different signalling networks via coupling to distinct G proteins. Thus global effects regulated by both receptors in events such as cardiac hypertrophy should be assessed independently. We first demonstrated distinct signalling pathway responses activated by the α1-AR compared to the ETR in hypertrophic cardiomyocytes. The upregulation of CREM expression after 1.5 h stimulation of the α1-AR suggested a concomitant increase in cAMP levels following receptor activation, as some CREM isoforms are upregulated in response to cAMP [56, 57]. We explored this potential signalling pathway further as upregulation of cAMP synthesis by catecholamines, the endogenous ligands for α1-AR, is usually associated with βAR receptor activation. We used a heterologous expression system with a panel of FRET- and BRET-based biosensors in HEK 293SL cells and the pertinent subtypes of the α1-AR and ETR that regulate cardiac hypertrophy. Whereas ETAR stimulation did not increase cAMP production or PKA activity, both α1A-AR and α1B-AR were able to generate cellular cAMP accumulation and PKA activation in a Gαs-dependent manner. This expands the current view of the α1-AR subfamily, which is classically associated with Gαq, to include regulation of cAMP and PKA through Gαs. Previous studies have demonstrated the ability of the α1-AR to lead to accumulation of cAMP through different mechanisms. Such increases were found to be secondary to activation of protein kinase C [58, 59] or through direct activation of Gαs [27, 28, 30, 60]. These studies assessed cAMP production in the whole cells and determined signalling pathways using small molecule inhibitors or co-immunoprecipitation of G proteins. Here we show directly that the increase in cAMP downstream of the α1-AR is dependent on the presence of Gαs. On the other hand, heterologous ETAR expression in Chinese hamster ovary cells showed the ability of the receptor to activate Gαs [61, 62], whereas ETAR activated PKA in a cAMP-independent manner in HeLa cells [63]. We have demonstrated that in HEK 293, ETAR does not activate PKA, either in a Gαs-dependent or -independent manner. Therefore, depending on the cellular context and the complements of G proteins, ETAR may be able to functionally couple to distinct signalling pathways. Increases in α1-AR densities have been noted during the progression to heart failure, especially in patients treated with β-blocking agents [64, 65]. This leads to an increase of the α1-AR to βAR ratio. It has been suggested that α1-AR may therefore assume a greater functional role in the failing heart by acting as a secondary inotropic system when β-adrenergic signalling is compromised by drugs or downregulation of βAR. Although we observed increases in α1-AR mediated cAMP production separately in the nucleus and cytoplasm, compartment specificity was observed for PKA activation. GPCRs and their effector proteins are commonly found in multiprotein signalosomes with A-kinase anchoring proteins (AKAPs) serving as scaffolds [66]. These AKAPs bring the components of signalling cascades into close proximity with one another, including the GPCR, adenylyl cyclases, cAMP phosphodiesterases, PKA, as well as different substrates [67, 68]. These complexes contain both positive and negative regulators of cAMP synthesis, which allows for discrete localized signalling and activation of specifically-localized subsets of PKA near their substrates [69]. PKA substrate phosphorylation following α1-AR stimulation was observed to be highly compartmentalized within the cell, and was delocalized by microtubule disruption [27]. More recently, an AKAP-Lbc signaling complex was shown to regulate α1-AR signalling through RhoA [70]. The nuclear-specific activation of PKA by the α1A-AR, despite cAMP production in both the cytoplasm and nucleus suggests interaction with different AKAP complexes. Multiple AKAPs have been shown to interact with the same GPCR. For example, the βAR interacts with both AKAP250 and AKAP150. To determine the compartment-specific AKAP interactions, future experiments with isoform specific AKAP disrupting peptides could be performed [71].