Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCRs oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.
BackgroundRecent studies indicate that α1‐adrenergic receptors (α1‐ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G‐protein‐coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous α1‐ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of α1‐ARs with their physiologic function by examining α1‐AR‐induced contractility in adult cardiac myocytes.Methods and ResultsBy measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the α1A‐subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an α1A‐subtype nuclear localization mutant in cardiac myocytes lacking α1‐ARs failed to rescue nuclear α1A‐mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked α1A‐mediated phosphorylation of cTnI. These data indicate that α1‐AR signaling originates in the nucleus. Consistent with these observations, we localized the α1A‐subtype to the inner nuclear membrane, identified PKCα, δ, and ε in the nucleus, and found that α1‐ARs activate PKCδ in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCδ nuclear localization mutant blunted α1‐induced phosphorylation of cTnI.ConclusionsTogether, our data identify a novel, “inside‐out” nuclear α1A‐subtype/PKCδ/cTnI‐signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of α1‐ARs and α1‐AR‐mediated physiologic function.
G protein-coupled receptors that signal through Gα (G receptors), such as α-adrenergic receptors (α-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cβ1 (PLCβ1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP) to produce inositol 1,4,5-trisphosphate (IP) and diacylglycerol. Despite these common proximal signaling mechanisms, G receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, G receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation-transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction IP-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of G-receptor signaling as a mechanistic basis for unique G receptor-induced hypertrophic phenotypes in cardiac myocytes. We show that α-ARs co-localize with PLCβ1 and PIP at the nuclear membrane. Further, nuclear α-ARs induced intranuclear PLCβ1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response ( significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCβ1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α-AR-induced transcription. In summary, these results link G-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation-transcription coupling in adult cardiac myocytes that accounts for differential G-receptor localization and better explains distinct physiological functions of G receptors.
In adult cardiac myocytes, multiple G protein-coupled receptors (GPCR) localize to and signal at the nucleus. These include endothelin B receptors, angiotensin type 1 and 2 receptors, β1- and β3-adrenergic receptors, and α1A- and α1B-adrenergic receptors. Initiation of signaling through nuclear GPCRs requires that ligands be produced within or transported into the cardiac myocytes, yet mechanisms whereby these ligands are produced or transported into cardiac myocytes are largely unclear. To activate nuclear adrenergic receptors in adult cardiac myocytes, uptake of endogenous catecholamines epinephrine and norepinephrine occurs via organic cation transporter 3 (OCT3), a member of the slc22a family of genes. This chapter details a method to detect and quantify catecholamine uptake in intact adult cardiac myocytes using a fluorescent-based catecholamine uptake assay.
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