Agonist-independent Nuclear Localization of the Apelin, Angiotensin AT1, and Bradykinin B2 Receptors

Abstract

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT 1 and bradykinin B 2 receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT 1 and B 2 receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellularfractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT 1 , and B 2 receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT 1 , and B 2 receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.The apelin receptor was discovered as an orphan G proteincoupled receptor (GPCR) 1 known as APJ, sharing highest identity with the angiotensin II AT 1 receptor, although no binding to the receptor was observed with angiotensin II (1). The apelin peptide was subsequently discovered as the endogenous ligand for this receptor (2). At least two isoforms of the apelin peptide are known to exist, apelin-36 and apelin-13, both of which act as agonists of the apelin receptor with distinct pharmacological properties (2-4). The apelinergic system has a widespread pattern of distribution in the brain and periphery as shown for the apelin peptide (3, 5-8) and receptor (6, 9 -11). Apelin has been shown to lower blood pressure (6, 12) and modulate contractility of cardiac tissue and blood vessels (13, 14), pituitary hormone release (15), fluid consumption (6, 15, 16), and cytokine suppression (17) and may have a role as a co-receptor for human immunodeficiency virus entry into cells (18,19). Overall, the apelinergic system is most closely related to the angiotensin and bradykinin systems, as observed through peptide and receptor structural and sequence similarities, expression patterns, and physiolo...

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“…This receptor is 380 amino acids long, consists of seven transmembrane domains and also includes a signal sequence that allows agonist-independent nuclear localization, a feature that may be cell-specific [27]. GPCRs are the estimated targets of nearly half of all currently available clinically used drugs [11] and are key components of the signal transduction machinery [35].…”

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

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“…ETB contains a sequence of basic amino acids (KRFK) in the eighth helix that could serve as a classical nuclear localization sequence [44,45]. Most proteins containing this type of sequence are transported from the cytoplasm into the nucleus by the importin α/β heterodimer [46].…”

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