Membrane-permeable tastants amplify β2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2's kinase activity

Einav Malach, Merav E. Shaul, Irena Peri, Liquan Huang, Andrew Spielman, Rony Seger, Michael Naim

Research output: Contribution to journalArticle

Abstract

Background Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methods β<inf>2</inf>AR-transfected cells and cells containing endogenous β<inf>2</inf>AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β<inf>2</inf>AR phosphorylation and β<inf>2</inf>AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β<inf>2</inf>AR phosphorylation and internalization. Results Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β<inf>2</inf>ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β<inf>2</inf>AR internalization. Conclusion Amphipathic tastants significantly amplify β<inf>2</inf>AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General Significance Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2's kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.

Original languageEnglish (US)
Pages (from-to)1375-1388
Number of pages14
JournalBBA - General Subjects
Volume1850
Issue number7
DOIs
StatePublished - Jul 1 2015

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Isoproterenol
Adrenergic Receptors
Phosphorylation
Phosphotransferases
Membranes
Amplification
1-Methyl-3-isobutylxanthine
Caffeine
Protein Kinases

Keywords

  • Desensitization
  • GRK2
  • Membrane-permeable
  • Signaling
  • Tastants
  • β<inf>2</inf>AR

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Membrane-permeable tastants amplify β2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2's kinase activity. / Malach, Einav; Shaul, Merav E.; Peri, Irena; Huang, Liquan; Spielman, Andrew; Seger, Rony; Naim, Michael.

In: BBA - General Subjects, Vol. 1850, No. 7, 01.07.2015, p. 1375-1388.

Research output: Contribution to journalArticle

Malach, Einav ; Shaul, Merav E. ; Peri, Irena ; Huang, Liquan ; Spielman, Andrew ; Seger, Rony ; Naim, Michael. / Membrane-permeable tastants amplify β2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2's kinase activity. In: BBA - General Subjects. 2015 ; Vol. 1850, No. 7. pp. 1375-1388.
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abstract = "Background Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methods β2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization. Results Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization. Conclusion Amphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General Significance Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2's kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.",
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T1 - Membrane-permeable tastants amplify β2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2's kinase activity

AU - Malach, Einav

AU - Shaul, Merav E.

AU - Peri, Irena

AU - Huang, Liquan

AU - Spielman, Andrew

AU - Seger, Rony

AU - Naim, Michael

PY - 2015/7/1

Y1 - 2015/7/1

N2 - Background Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methods β2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization. Results Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization. Conclusion Amphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General Significance Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2's kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.

AB - Background Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. Methods β2AR-transfected cells and cells containing endogenous β2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, β2AR phosphorylation and β2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of β2AR phosphorylation and internalization. Results Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of β2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated β2AR internalization. Conclusion Amphipathic tastants significantly amplify β2AR signaling and delay its desensitization via their intracellular inhibition of GRK2. General Significance Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2's kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.

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KW - Membrane-permeable

KW - Signaling

KW - Tastants

KW - β<inf>2</inf>AR

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