Elsevier

Cellular Signalling

Volume 24, Issue 3, March 2012, Pages 794-802
Cellular Signalling

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

https://doi.org/10.1016/j.cellsig.2011.11.014Get rights and content

Abstract

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 GPCR 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.

Highlights

► Endogenous α1-ARs localize to the nucleus in adult cardiac myocytes. ► Nuclear localization sequences drive α1-ARs to the nuclei in adult cardiac myocytes. ► α1-AR signaling is initiated in the nucleus in adult cardiac myocytes. ► α1-ARs form oligomers that alter receptor function in adult cardiac myocytes.

Introduction

Conventional models of G-protein coupled receptor (GPCR) signaling describe receptor activation at the plasma membrane leading to initiation of downstream signaling within the cell commonly referred to as “outside-in” signaling. However, it has recently become clear that a number of GPCRs localize to and signal at the nuclear membrane in a variety of cell types including neurons, hepatocytes, and cardiac myocytes (reviews [1], [2], [3]). Endothelin receptors, angiotensin receptors, and β-adrenergic receptors (β-ARs) were all recently reported to localize to the nuclei in adult cardiac myocytes (cardiac myocytes in most species have two nuclei) [4], [5], [6], [7]. However, each of these receptors also localizes to the plasma membrane, therefore the functional significance of localization to the nuclei in adult cardiac myocytes remains unclear. Yet, studies using nuclei isolated from adult cardiac myocytes indicated that endothelin receptors induced calcium transients [4], and β-ARs and angiotensin receptors increased gene transcription, suggesting a potential physiologic significance for nuclear GPCR signaling [6], [7].

Targeting of proteins to the nucleus involves nuclear localization sequences embedded in the protein, which consist of mono- or bi-partite basic residues, usually lysines and arginines, or glycine-arginine repeats [8], [9], [10]. A family of proteins called importins binds to nuclear localization sequences and facilitates localization to the nucleus. This importin-mediated nuclear localization occurs for proteins that target to the nucleoplasm as well as the inner nuclear membrane [11], [12], [13]. This method of nuclear localization was previously described for the type 1 parathyroid hormone receptor [14], [15] and more recently for the gonadotropin-releasing hormone type 1 receptor [16].

Another newly described function of GPCRs is the ability to form oligomers, and receptor hetero-oligomers have been proposed to affect receptor ligand binding, expression, internalization, and signaling. To date, several GPCRs have been shown to form homo- and hetero-oligomers that alter receptor function, including opioid receptors, dopamine receptors, adenosine receptors, angiotensin receptors, and β-ARs (reviews [17], [18], [19], [20]). In HEK293 cells, α1-adrenergic receptors (α1-ARs) formed homo- and hetero-oligomers, demonstrating the capacity for α1-ARs to form oligomers [21], [22], [23], [24]. Moreover, α1A- and α1B-subtype hetero-oligomers altered receptor internalization [23], and α1B- and α1D-subtype hetero-oligomers were required for cell surface expression of the α1D-subtype in HEK293 cells [22]. In cardiac myocytes, β1- and β2-ARs formed hetero-oligomers that altered β-AR mediated contractile function, suggesting the possibility that adrenergic receptor oligomers could affect cardiac myocyte function [25].

In general, the lack of validated α1-AR subtype-specific antibodies [26] has hindered studies attempting to define α1-AR subcellular localization. However, we overcame this obstacle by using radio-ligand binding assays on fractionated cardiac myocytes and a fluorescent α1-AR ligand to label receptors in cultured cardiac myocytes, and we localized endogenous α1-ARs to the nuclei in adult cardiac myocytes [27]. Further, we demonstrated that the downstream α1-AR signaling partners Gαq and phospholipase Cβ1 co-localized with α1-ARs only at the nuclei in adult cardiac myocytes [27]. We also developed a reconstitution system in α1A- and α1B-AR double knockout (α1ABKO) cardiac myocytes, which lack endogenous α1-ARs, using α1A- and α1B-GFP fusion proteins that recapitulated the localization of the two endogenous α1-subtypes at the nuclei [27]. Finally, we found that organic cation transporter-3 (OCT3) facilitated rapid uptake of catecholamines into adult cardiac myocytes and that this uptake was required for α1-AR signaling [27]. In short, our data indicated that endogenous α1-ARs localize to and initiate signaling at the nucleus in adult cardiac myocytes.

In this report, we examined α1-AR expression in nuclei isolated from adult cardiac myocytes and validated that endogenous α1-ARs localized to the nuclei in adult cardiac myocytes and that our α1-GFP constructs reproduced this localization. We identified putative nuclear localization sequences in both the α1A- and α1B-subtypes and demonstrated that α1-AR nuclear localization was required for signaling. Furthermore, we demonstrated that co-localization of the α1A- and α1B-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could regulate receptor signaling in adult cardiac myocytes. Together, our data demonstrate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.

Section snippets

Mice

The generation of α1A- (α1AKO) and α1B- (α1BKO) AR single knockout, as well as α1A-/α1B- (α1ABKO) AR double knockout mice, was previously described [28]. 12th–15th generation congenic C57BL/6J mice between 10 and 15 weeks of age were used in all experiments. The use of all animals in this study conformed to the PHS Guide for Care and Use of Laboratory Animals and was approved by Sanford Research/USD Institutional Animal Care and Use Committee.

Reagents

All reagents were made using chemicals purchased from

Endogenous α1-ARs localize to the nuclei in adult cardiac myocytes

Here, we set out to validate and extend our previous result that endogenous α1-ARs localize to the nuclei in adult cardiac myocytes [27]. Intact nuclei were fractionated from freshly isolated wild-type adult mouse cardiac myocytes by sucrose density gradient ultracentrifugation. Endogenous α1-ARs were detected on the nuclear membrane with the fluorescently labeled α1-antagonist BODIPY-prazosin counterstained with the DNA stain Hoescht 33342 to identify nuclei (Fig. 1A). We previously

Discussion

Here, we examined α1-AR localization to the nuclei in adult cardiac myocytes, the mechanism behind α1-AR nuclear localization, and how nuclear localization affected receptor function. In short, we validated our previous findings, clearly demonstrating that endogenous α1-ARs localized to the nuclear membrane in adult cardiac myocytes. Furthermore, we identified functional nuclear localization sequences in both the α1A- and α1B-subtypes that drove nuclear localization and demonstrated that

Acknowledgments

This work was supported by grants from the National Institute of Health (P20 RR-017662, TDO; F32 HL085980-02, CDW). The authors would also like to thank Chastity L. Healy, Kelly Graber of the Sanford Research Imaging Core, Andy Cypher of the Sanford Research Cell Culture Core, and Yuan Huang, MD of the Sanford Research Molecular Biology Core for their excellent technical assistance. The Sanford Research Cores listed here were supported by a grant from the National Institutes of Health (P20

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    These authors contributed equally to this work.

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