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Carboxyl terminus-truncated ?1D-adrenoceptors inhibit the ERK pathway.

ABSTRACT: Human ?1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human ?1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states. Amino terminus-truncated and both amino and carboxyl termini-truncated ?1D-adrenoceptors were transfected into Rat-1, HEK293, and B103 cells, and changes in the phosphorylation state of extracellular signal-regulated kinase was assessed using biochemical and biophysical approaches. The phosphorylation state of other protein kinases (p38, MEK1, and Raf-1) was also studied. Noradrenaline-induced ERK phosphorylation in Rat-1 fibroblasts expressing amino termini-truncated ?1D-adrenoceptors. However, in cells expressing receptors with both amino and carboxyl termini truncations, noradrenaline-induced activation was abrogated. Interestingly, ERK phosphorylation that normally occurs through activation of endogenous G protein-coupled receptors, EGF receptors, and protein kinase C, was also decreased, suggesting that downstream steps in the mitogen-activated protein kinase pathway were affected. A similar effect was observed in B103 cells but not in HEK 293 cells. Phosphorylation of Raf-1 and MEK1 was also diminished in Rat-1 fibroblasts expressing amino- and carboxyl-truncated ?1D-adrenoceptors. Our data indicate that expression of carboxyl terminus-truncated ?1D-adrenoceptors alters ERK and p38 phosphorylation state.

SUBMITTER: Alfonzo-Mendez MA

PROVIDER: S-EPMC6042655 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway.

Alfonzo-Méndez Marco A MA Castillo-Badillo Jean A JA Romero-Ávila M Teresa MT Rivera Richard R Chun Jerold J García-Sáinz J Adolfo JA

Naunyn-Schmiedeberg's archives of pharmacology 20160505 8

Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states. Amino terminus-truncated and both amino and carboxyl termini-truncated α1D-adrenoceptors were transfect ...[more]

PMID: 27146292

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Project description:Background ? Carboxyl terminus 1 (?CT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that ?CT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase C? phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of ?CT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study ?CT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase C? phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the ?CT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an ?-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that ?CT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of ?CT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of ?CT1 (?CT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of ?CT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

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Carboxyl terminus-truncated ?1D-adrenoceptors inhibit the ERK pathway.

Publications

Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway.

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