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  • Two GPCR subtypes of interest in the


    Two GPCR subtypes of interest in the myocardium are the endothelin receptor (ETR) and the α1-adrenergic receptor (α1-AR). Upon ligand binding, these receptors canonically activate the Gαq protein leading to activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3), and a subsequent increase in intracellular Ca2+ levels and protein kinase C (PKC) activation. ETR and α1-AR, in response to endothelins or the endogenous catecholamines Guvacoline hydrobromide sale and norepinephrine respectively, mediate signalling events important for cardiac function and pathology (reviewed in [3]). The ETR family contains two subtypes, ETA and ETB, that are expressed at similar levels in the heart [[4], [5], [6], [7]]. ETR subtypes are able to regulate multiple signalling pathways including phospholipase D, phospholipase A2, Na+/H+ exchangers, cAMP and cGMP production, mitogen activated protein kinase (MAPK) pathways, and tyrosine kinases [[8], [9], [10], [11], [12], [13], [14], [15]]. In the heart, ETR signalling has inotropic and chronotropic effects [16, 17] and mediates cardiac remodeling in hypertrophy, myocardial infarction, and congestive heart failure [[18], [19], [20]]. In these various cardiac pathologies, ETR signalling through endothelin-1 is increased and the associated cardiac hypertrophy can be blocked with an ETAR-specific antagonist [[21], [22], [23], [24]]. The α1-AR family consists of multiple subtypes, including the α1A-, α1B- and α1D-ARs [25, 26]. A role for α1-ARs has been demonstrated in the regulation of phospholipase D, phospholipase A2, MAPK pathways, Na+/H+ exchangers, tyrosine kinases, as well as cAMP and cGMP production [10, 14, 15, [27], [28], [29], [30], [31], [32]]. Cardiac α1-ARs have some inotropic and chronotropic effects and also regulate cardiac remodeling in hypertrophy and following myocardial infarctions (reviewed in [33, 34]). All three subtypes are expressed in cardiomyocytes but only α1A-AR and α1B-AR are detectable at the protein level [35]. Differences between the receptor subtypes in mediating cardiac pathologies have been identified through the use of transgenic mice. Cardiac specific overexpression of α1B-AR led to an exacerbated hypertrophic response to pressure overload and dilated cardiomyopathy, whereas overexpression of α1A-AR did not affect the response [[36], [37], [38], [39]]. Initially, studies of GPCRs predominantly assessed the signalling pathways downstream of receptors on the cell surface. There is now an understanding that GPCRs can localize to and signal from various intracellular compartments, such as the nucleus (reviewed in [40]). These intracellular pools of receptors can lead to distinct signalling pathways from those activated by the same receptor at the cellular surface. In adult rat ventricular cardiomyocytes, ETBR localizes along the nuclear membrane, whereas ETAR is mainly found at the cell surface [41]. In the α1-AR family, both subtypes found in adult cardiomyocytes localize to the nucleus and to a lesser extent the cell surface [42]. The nuclear GPCR population activates proximal signalling pathways similar to those on the cell membrane, but also have more direct effects on nuclear activities such as transcription initiation and gene expression (reviewed in [40, 43]). Studies assessing these nuclear specific events have used both the ETR and α1-AR interchangeably as both are thought to predominantly couple to Gαq. Furthermore, the receptor subtype-specific signalling that occurs in distinct cellular compartments has not been addressed. 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 bioluminescent 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.