Although we observed increases in AR mediated
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 . 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 . PKA substrate phosphorylation following α1-AR stimulation was observed to be highly compartmentalized within the cell, and was delocalized by microtubule disruption . More recently, an AKAP-Lbc signaling complex was shown to regulate α1-AR signalling through RhoA . The nuclear-specific activation of PKA by the α1A-AR, despite cAMP production in both the you can check here 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 .
PKA signalling in the nucleus was thought to be due to the translocation of the catalytic subunit upon activation from the cytoplasm to the nucleus via diffusion . However, a new understanding has emerged, as both the regulatory and catalytic subunits have been identified in the nucleus and functionally separate from the cell surface [, , ]. Functional differences between the two pools of PKA have been identified in cardiomyocytes, with cytoplasmic PKA exerting inotropic effects and the nuclear pool regulating hypertrophic responses . The compartment specific activation of PKA by different subtypes of the α1-AR adds another dimension to their differential physiological and pathological roles. Subtype selective agonists or antagonists could be used to assess these differences in cardiomyocytes.
Conclusion In conclusion, we have provided evidence that the α1-AR family activates the cAMP/PKA pathway in a Gαs-dependent manner. Within this subfamily, there is subtype specific activation of PKA in various cellular compartments. Furthermore, the inability of the ETAR to activate PKA highlights that when studying global effects in cardiac hypertrophy, agonists for GPCRs that canonically couple to Gαq need to be assessed independently for additional signalling phenotypes.
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Acknowledgements This work was supported by a grant from the Heart and Stroke Foundation of Canada to TEH and JCT. RDM was supported by a scholarship from the Canadian Institutes of Health Research (CIHR). JCT was supported by a fellowship from Fonds de recherche du Québec Santé (FRQS). RDM and NA were supported by a fellowship and studentship, respectively, from the McGill-CIHR Drug Development Training Program and from the Mathematics of Information Technology and Complex Systems (MITACS). KB was supported by a Faculty of Medicine Doctoral Scholarship and YS received a summer bursary from the Groupe d\'étude des protéines membranaires (GEPROM). The authors thank Viviane Pagé for administrative and technical support.
Over the years, a number of studies have reported results concerning the behavior of the two endothelin receptor subtypes, ETA and ETB, that do not fit the classical model of two G protein-coupled receptors acting independently of one another. For example, in the rat anterior pituitary gland, both ETA and ETB receptors are expressed, but competitive binding studies indicated that ETB receptor-selective ligands competed for binding with endothelin-1 (ET-1) only when an ETA receptor-selective ligand was present. Simultaneous blockade of both receptor subtypes was necessary to inhibit clearance of ET-1 by astrocytes, with antagonists of individual receptor subtypes having no effect when administered alone. A similar cooperative interaction between the two receptors, necessitating blockade of both subtypes in order to abolish responses to ET-1, has also been noted in a variety of preparations involving vascular or airway smooth muscle. Systematic experiments by Just and colleagues demonstrated that the contributions of each receptor subtype to the renal vascular effects of ET-1 appear highly complex and are not merely additive. This may not be surprising given differences in receptor expression along the renal vascular tree combined with vasodilator and ET-1 clearance functions of endothelial-cell ETB receptors. However, those factors do not adequately explain the observation that either an ETA receptor-selective or an ETB receptor-selective antagonist was able to abolish afferent arteriolar constrictor responses to low concentrations of ET-1.