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

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 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.

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

“…Hence, it is unlikely that the presence of ETB on nuclear membranes is a result of 'overflow' from the endoplasmic reticulum. Interestingly, both ETA and ETB, along with several other GPCRs, possess a nuclear localization sequence within their carboxyl tail region [44,59]. The presence of a similar nuclear localization sequence in both ETA and ETB implies the presence of additional structural determinants that either direct ETB to segregate to the nucleus or ETA to the plasma membrane.…”

“…The presence of a similar nuclear localization sequence in both ETA and ETB implies the presence of additional structural determinants that either direct ETB to segregate to the nucleus or ETA to the plasma membrane. Both α1A-and α1B-adrenergic receptors are found on nuclear membranes in ventricular myocytes [59,60] and both were recently shown to possess nuclear localization sequence motifs within their C-terminal domains, although, interestingly, not the same type of nuclear localization sequence [59]. In either case, site-directed mutations within this sequence prevented sorting of either receptor to the nucleus, but did not result in their relocalization to the plasma membrane [59].…”

“…Both α1A-and α1B-adrenergic receptors are found on nuclear membranes in ventricular myocytes [59,60] and both were recently shown to possess nuclear localization sequence motifs within their C-terminal domains, although, interestingly, not the same type of nuclear localization sequence [59]. In either case, site-directed mutations within this sequence prevented sorting of either receptor to the nucleus, but did not result in their relocalization to the plasma membrane [59]. Furthermore, when stably expressed in HEK293 cells, ETB localizes primarily to the plasma membrane [61] (Merlen and Allen, unpublished).…”

Intranuclear Signaling Cascades Triggered by Nuclear GPCRs

“…The α1A and β1B subtypes co-localize at the nucleus with the formation of receptor oligomers that affect receptor signaling [27], but no direct demonstration has been provided for interaction between β1-AR and β3-AR in the nucleus (Table 1) [21]. In the two α1-AR subtypes has been identified a bipartite NLS in α1A and a glycine-arginine repeat/ arginine rich NLS in the α1B, in the carboxyl tail region of the respective receptors (Table 2) [28].…”

Caged ligands to study the role of intracellular GPCRs

“…In contrast, PGE 2 and catecholamines are taken up by specific transporters [5][6][7]. In both neonatal and adult cardiomyocytes, the effects of extracellular phenylephrine are mediated, in part, by intracellular α 1 AR [7][8][9] and extracellularly applied [ 3 H]norepinephrine accumulates within the nuclei of neonatal ventricular cardiomyocytes [10]. Hence, hydrophobic ligands that are able to cross the plasma membrane may show no selectivity for activating cell surface or intracellular signalling.…”