Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades

Garnovskaya, Maria N.; Mukhin, Y.; Raymond, Roger

doi:10.1042/bj3300489

Cited by 43 publications

(55 citation statements)

References 67 publications

(91 reference statements)

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“…In CHO-K1 cells, the 5-HT 1A receptor rapidly activates Erk via a pathway that involves pertussis toxin-sensitive G protein ␤␥ subunits, phosphatidylinositol 3Ј-kinase and src kinase, the Shc and Grb2 adapter proteins, mSos, Ras, and Raf (13,15 lyl cyclase and G proteins in clathrin-rich brain vesicles (16). In this report, we show that 5-HT 1A receptor-mediated Erk activation is sensitive to inhibitors of Ca 2ϩ /CAM and clathrinmediated endocytosis but not to inhibitors of the known CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterases.…”

Section: ؉mentioning

confidence: 99%

“…Monolayers were then lysed directly using 200 l/well Laemmli sample buffer. Phosphorylation of Erk1/2 was detected by immunoblotting using rabbit phosphospecific Erk IgG (New England Biolabs) as described previously, except that the bands were visualized with Vistra ECF reagent (Amersham Pharmacia Biotech) (13,15). Membranes were stripped and reprobed with rabbit anti-Erk2 IgG (Santa Cruz Biotechnology) to quantitate total Erk2.…”

Section: Materials-12-bis(2-aminophenoxy)ethane-nnnјnј-tetraaceticmentioning

confidence: 99%

“…CHO-1A-27 cells (18) were maintained in Ham's F-12 nutrient mixture (Life Technologies, Inc.) supplemented with 10% FBS, 50 g/ml gentamicin, and 400 g/ml G418. Transfections were performed on 80 -90% confluent monolayers in 100-mm dishes, using calcium phosphate coprecipitation for HEK-293 cells (23) and LipofectAMINE for CHO cells (9 l/g of DNA, Life Technologies, Inc.) (15). Empty pcDNA3 vector was added to transfections to keep the total mass of DNA added per dish constant within experiments.…”

Section: Materials-12-bis(2-aminophenoxy)ethane-nnnјnј-tetraaceticmentioning

confidence: 99%

“…In CHO-K1 cells, the 5-HT 1A receptor rapidly activates Erk via a pathway that involves pertussis toxin-sensitive G protein ␤␥ subunits, phosphatidylinositol 3Ј-kinase and src kinase, the Shc and Grb2 adapter proteins, mSos, Ras, and Raf (13,15). Moreover, 5-HT 1A receptors have been colocalized with adeny-lyl cyclase and G proteins in clathrin-rich brain vesicles (16).…”

mentioning

confidence: 99%

“…Immune complexes were washed twice in icecold RIPA buffer and twice in kinase buffer (50 mM HEPES, pH 7.0, 10 mM MgCl 2 , 1 mM dithiothreitol). Phosphorylation assays were performed as described previously using myelin basic protein as the substrate (6,13,15). Both assays of ERK activity yielded virtually identical results.…”

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confidence: 99%

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Serotonin 5-HT1A Receptor-mediated Erk Activation Requires Calcium/Calmodulin-dependent Receptor Endocytosis

Rocca

Mukhin

Garnovskaya

et al. 1999

Journal of Biological Chemistry

145

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Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The G i -coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT 1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrinmediated endocytosis. This activation was attenuated by chelation of intracellular Ca 2؉ and Ca 2؉/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT 1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT 1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestrationdefective mutants of ␤-arrestin 1 and dynamin attenuated 5-HT 1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT 1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT 1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.Receptors coupled to heterotrimeric guanine nucleotidebinding (G) proteins, the largest known family of cell surface receptors, mediate cellular responses to many extracellular stimuli, such as neurotransmitters, peptide hormones, odorants, and photons (1). In addition to regulating the generation of soluble second messengers, many G protein-coupled receptors mediate proliferative or differentiative signals in various cultured cell lines and tissues via mitogen-activated protein (MAP) 1 kinases (2, 3). Erk1/2 MAP kinases are serine/threonine kinases involved in the activation of nuclear transcription factors. Erk1/2 activity is regulated by threonine/tyrosine phosphorylation, which is controlled by a highly conserved phosphorylation cascade. Phosphorylation of Erk1/2 is catalyzed by the MAP/Erk kinases 1 and 2 (MEK1 and MEK2) that are themselves phosphorylated and activated by MEK kinases such as raf-1 oncogene family proteins. Activation of Raf-1 occurs as a consequence of membrane translocation, which can be mediated by the GTP-bound form of the small G protein, Ras.Like the epidermal growth factor (EGF) receptor and other receptor tyrosine kinases, many G protein-coupled receptors regulate Ras function via tyrosine phosphorylation. Several G i -coupled receptors stimulate pertussis toxin-sensit...

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Section: ؉mentioning

confidence: 99%

Section: Materials-12-bis(2-aminophenoxy)ethane-nnnјnј-tetraaceticmentioning

confidence: 99%

Section: Materials-12-bis(2-aminophenoxy)ethane-nnnјnј-tetraaceticmentioning

confidence: 99%

“…In CHO-K1 cells, the 5-HT 1A receptor rapidly activates Erk via a pathway that involves pertussis toxin-sensitive G protein ␤␥ subunits, phosphatidylinositol 3Ј-kinase and src kinase, the Shc and Grb2 adapter proteins, mSos, Ras, and Raf (13,15). Moreover, 5-HT 1A receptors have been colocalized with adeny-lyl cyclase and G proteins in clathrin-rich brain vesicles (16).…”

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confidence: 99%

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confidence: 99%

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