Subcellular Compartmentalization of Proximal G Q
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ARTICLE cro ␣ Subcellular compartmentalization of proximal G q-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes Received for publication, February 6, 2018, and in revised form, March 13, 2018 Published, Papers in Press, April 2, 2018, DOI 10.1074/jbc.RA118.002283 Erika F. Dahl‡, Steven C. Wu§, Chastity L. Healy§, Brian A. Harsch¶, Gregory C. Shearer¶, and Timothy D. O’Connell§1 From the Departments of ‡Pharmacology and §Integrative Biology and Physiology, University of Minnesota, Minneapolis, Minnesota 55455 and the ¶Department of Nutritional Sciences, Pennsylvania State University, University Park, Pennsylvania 16802 Edited by Henrik G. Dohlman ␣ ␣ G protein–coupled receptors that signal through G q (Gq G protein–coupled receptors that signal through G q (Gq ␣ ␣ 2 receptors), such as 1-adrenergic receptors ( 1-ARs) or angio- receptors) share a common proximal signaling pathway Downloaded from tensin receptors, share a common proximal signaling pathway through the activation of phospholipase C1 (PLC1), which   that activates phospholipase C 1 (PLC 1), which cleaves phos- cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to pro- phatidylinositol 4,5-bisphosphate (PIP2) to produce inositol duce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol 1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these com- (DAG) (1). However, in any cell, simultaneous activation of mon proximal signaling mechanisms, Gq receptors produce dis- multiple proximal Gq-receptor signaling events would preclude http://www.jbc.org/ tinct physiological responses, yet the mechanistic basis for this the cell’s ability to process different signals and produce a unique outcome. Despite this commonality, each G receptor remains unclear. In the heart, Gq receptors are thought to q induce myocyte hypertrophy through a mechanism termed mediates distinct physiologic processes, but how this specificity excitation–transcription coupling, which provides a mechanis- is achieved is unclear in many cases. Compartmentalization of tic basis for compartmentalization of calcium required for con- signaling provides one mechanism through which a cell could traction versus IP -dependent intranuclear calcium required for handle a multitude of potentially overlapping signals. Cardiac at University of Minnesota Libraries on June 8, 2018 3 myocytes offer several examples of compartmentalized recep- hypertrophy. Here, we identified subcellular compartmental- tor signaling (e.g. the ability to discriminate -adrenergic ization of G -receptor signaling as a mechanistic basis for q (-AR)-G -induced calcium signals that augment contractility unique G receptor–induced hypertrophic phenotypes in car- s q from G receptor–mediated calcium signals that induce hyper- diac myocytes. We show that ␣ -ARs co-localize with PLC1 q 1 trophy) (2). In this case, inositol-sensitive calcium release ␣ and PIP2 at the nuclear membrane. Further, nuclear 1-ARs occurs in the nucleus based on the localization of the inositol  induced intranuclear PLC 1 activity, leading to histone trisphosphate receptor to the inner nuclear membrane. How- deacetylase 5 (HDAC5) export and a robust transcriptional ever, this ultimately raises questions about how Gq receptors, response (i.e. significant up- or down-regulation of 806 traditionally thought to localize to the cell surface, might acti- genes). Conversely, we found that angiotensin receptors vate a nuclear calcium signal to regulate hypertrophy. localize to the sarcolemma and induce sarcolemmal PLC1 ␣ In cardiac myocytes, Gq receptors, including 1-adrenergic, activity, but fail to promote HDAC5 nuclear export, while ␣ endothelin, and angiotensin receptors ( 1-AR, ET-R, and producing a transcriptional response that is mostly a subset AT-R, respectively), regulate vital signaling pathways control- ␣ of 1-AR–induced transcription. In summary, these results ling hypertrophy, cell survival, and inotropy that impact heart link Gq-receptor compartmentalization in cardiac myocytes failure (HF) (3). Early studies in cell and animal models estab- to unique hypertrophic transcription. They suggest a new lished the long-held convention that Gq signaling is maladap- model of excitation–transcription coupling in adult cardiac tive and exacerbates HF (4–6). However, several studies have myocytes that accounts for differential Gq-receptor localiza- challenged the convention that Gq-receptor signaling univer- tion and better explains distinct physiological functions of Gq sally worsens HF. Clinical studies indicate that antagonists tar- receptors. geting Gq receptors in human HF do not uniformly improve HF 2 The abbreviations used are: Gq receptor, Gq-coupled G-protein–coupled ␣ ␣ receptor; 1-AR, 1-adrenergic receptor; AMVM, adult mouse ventricular This work was supported by start-up funds from the University of Minnesota myocytes; AngII, angiotensin II; AT-R, angiotensin receptor type 1 and 2; (Minneapolis, MN) (to T. D. O.) and start-up funds from Pennsylvania State -AR, -adrenergic receptor; DAG, diacylglycerol; ET-R, endothelin recep- University (University Park, PA) (to G. C. S.). The authors declare that they tor; HF, heart failure; HDAC, histone deacetylase; IP, inositol phosphate; IP2, have no conflicts of interest with the contents of this article. inositol diphosphate; IP3, inositol-1,4,5-trisphosphate; IP3R, IP3 receptor; This article contains Tables S1–S4 and supporting information. NLS, nuclear localization sequence; OCT3, organic cation transporter 3; PE,  The RNA-Seq data set is available at ArrayExpress under accession number phenylephrine; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC 1, phos- E-MTAB-6770. pholipase C1; QDot, quantum dot; TAMRA, tetramethylrhodamine; 1 To whom correspondence should be addressed: Dept. of Integrative Biol- 2-APB, 2-aminoethoxydiphenyl borate; PC1 and PC2, principle component ogy and Physiology, University of Minnesota, 2231 6th St. SE, Cancer and 1 and 2, respectively; PHD, pleckstrin homology domain; ANOVA, analysis Cardiovascular Research Bldg., 3-141, Minneapolis, MN 55455. Tel.: 612- of variance; FPKM, fragments per kilobase of exon per million reads 625-6750; Fax: 612-301-1543; E-mail: [email protected]. mapped. 8734 J. Biol. Chem. (2018) 293(23) 8734–8749 © 2018 Dahl et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Compartmentalized myocyte Gq-receptor signaling outcomes. Although AT-R antagonists are standard of care (7), these compartmentalized proximal signals induce differential ␣ 1-AR antagonists worsen HF (3). In mice, 8-fold cardiac myo- activation of nuclear hypertrophic signaling pathways to pro- cyte-specific overexpression of Gq induces cardiac myocyte cell duce unique hypertrophic transcriptomes. Finally, these data death and HF (6). However, Gq levels are increased only 2-fold suggest an entirely new model of excitation–transcription cou- in human HF (8, 9), which in mice produces no obvious pheno- pling that accounts for differential localization of Gq receptors ␣ type (6). Our laboratory previously demonstrated that 1-ARs and better explains the distinct physiologic function of Gq are cardioprotective, as defined by their ability to initiate adapt- receptors in adult cardiac myocytes. ive hypertrophy, survival signaling, and positive inotropy ␣ (reviewed in Ref. 3). The finding that 1-ARs are cardioprotec- Results ␣ tive agrees with the clinical data indicating that 1-antagonists ␣ 1-ARs localize to the nuclei and AT-Rs localize to the worsen HF. Collectively, these data suggest that Gq receptors sarcolemma in adult cardiac myocytes ␣ are functionally unique, some protective, like 1-ARs, and oth- ers maladaptive, like AT-Rs. Here, we sought to define the subcellular localization of ␣ How can these apparently divergent data on cardiac G -re- 1-ARs and AT-Rs in adult cardiac myocytes (Figs. 1 and 2). q ␣ ceptor function be reconciled? A potentially important clue is Previously, we demonstrated that endogenous 1-ARs localize ␣ to the nucleus in adult mouse ventricular myocytes (AMVM) our finding that cardiac 1-ARs primarily localize to and signal Downloaded from at the nucleus unlike other G receptors (reviewed in Refs. 3 and (11, 12, 15). However, reagents typically employed to localize q ␣ 10). Using fluorescent ligands and binding assays in fraction- receptors are generally unreliable, especially 1-AR subtype– ated adult cardiac myocytes, we previously demonstrated that specific antibodies, which lack specificity (16), or fluorescent ␣ ligands, which are no longer commercially available, but none- endogenous 1-ARs localize primarily to the nucleus. We also found that ␣ -ARs contain nuclear localization sequences theless had suboptimal binding kinetics (12). To overcome 1 ␣ http://www.jbc.org/ ␣ these shortcomings, we developed a novel 1-AR ligand (NLSs), and that whereas reconstitution of WT 1-ARs in ␣ -knockout cardiac myocytes restores ␣ -signaling, reconsti- composed of 2-piperazinyl-4-amino-6,7-dimethoxyquinazo- 1 1 ␣ ␣ line, the common pharmacophore of 1-AR antagonists such as tution of 1-NLS mutant receptors does not, demonstrating a ␣ terazosin and doxazosin, attached to a PEG linker with a termi- requirement for 1-nuclear localization (11). Further, we ␣ nal biotin moiety fused to a streptavidin-coated fluorescent showed that 1-ARs activate intranuclear signaling in adult car- diac myocytes based on our observations that the ␣ -agonist quantum dot (QDot) with an emission wavelength