Abstract
G(q) protein-coupled receptor stimulation increases sarcolemmal Na(+)/H(+) exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A(1) receptor stimulation inhibits this response through a G(i) protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A(1) receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the alpha(1)-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A(c)) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G(i) proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A(c) and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A(c) dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A(1) receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G(i) protein-mediated translocation of PP2A(c) and NHE1 dephosphorylation.
Highlights
(1), which is one of nine known members of the solute carrier 9 (SLC9) gene family [2]
In adult rat ventricular myocytes (ARVM), stimulation of adenosine A1 receptors inhibits the increase in sarcolemmal NHE activity induced via Gq protein-coupled receptors, such as ␣1-ARs [27]
Taken together with the evidence presented above that ␣1-AR-mediated NHE1 phosphorylation is abolished by A1 receptor stimulation (Fig. 1B), these findings suggest the possible existence of two mechanisms, which are not mutually exclusive
Summary
This investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty’s Stationery Office, London. RSK2-mediated Phosphorylation of NHE1 in Vitro—The reaction mixture comprised recombinant active RSK2 (100 ng) and GST-NHE1 fusion protein (1 nmol) in TADB (containing 15 mmol/liter MgCl2), as well as unlabeled ATP (100 mol/ liter) with or without added [32P]ATP (1 Ci), and was incubated at 30 °C. Mediated phosphorylation was not seen by either method when GST was used as substrate (data not shown), confirming that the phosphorylation signal originated within the NHE1 domain of the fusion protein These data indicated that the phospho-Ser 14-3-3 binding motif antibody might be a useful tool for detecting RSK-mediated. A, phosphorylation of GST-NHE1 fusion protein by recombinant RSK2 in vitro, as detected by immunoblotting with a phospho-Ser 14-3-3 binding motif antibody or by 32P autoradiography.
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