Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: Evidence that transporter undergoes endoproteolytic cleavage

Дмитриев, А. Д.; Factor, M. I.; Segal, O.L.; Pavlova, Elena; Massino, Yu. S.; Smirnova, M. B.; Yakovleva, D. A.; Dmitriev, D. A.; Kizim, E. A.; Kolyaskina, G. I.; Os, Brusov

doi:10.1016/j.cccn.2004.12.019

Cited by 25 publications

(16 citation statements)

References 47 publications

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“…It has been recently demonstrated that SERT undergoes a deep proteolytic degradation during the extraction from cells, and that its degradation depends very much on the preparation conditions [43]. Accordingly, we have found that the usually employed anti-protease cocktails only slightly prevented the SERT proteolysis, which was considerably counteracted by high urea concentration.…”

Section: Discussionmentioning

confidence: 69%

Serotonin (5-HT) Transport in Human Platelets is Modulated by Src-Catalysed Tyr-Phosphorylation of the Plasma Membrane Transporter SERT

Zarpellon

Donella‐Deana²,

Folda³

et al. 2008

Cell Physiol Biochem

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Background/Aim: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. Methods: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. Results: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 κDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.

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Section: Discussionmentioning

confidence: 69%

Serotonin (5-HT) Transport in Human Platelets is Modulated by Src-Catalysed Tyr-Phosphorylation of the Plasma Membrane Transporter SERT

Zarpellon

Donella‐Deana²,

Folda³

et al. 2008

Cell Physiol Biochem

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“…Since the predicted molecular mass of SERT is 72 kDa [7], the finding of an abundant anti-SERT protein band of about 85 kDa suggested that the transporter was likely present in its glycosylated form [17,40]. On the other hand the anti-SERT bands displaying molecular masses lower than the expected 72 kDa indicated that the serotonin transporter likely underwent a proteolytic degradation during the experimental procedure, even in the presence of a protease inhibitor cocktail [41].…”

Section: Resultsmentioning

confidence: 99%

“…The major band displaying a Mr higher than that predicted of 72 kDa is likely due to glycosylation of the transporter [2,17]. Whereas the protein bands displaying Mr lower than 72 kDa most likely originated from a proteolytic process occurring during the SERT isolation procedure [41]. In fact we have found that SERT fragmentation increased by prolonging the precipitation time (from 2 h to overnight), while it decreased in the presence of high urea concentration (4 M) in the cellular disrupting medium (not shown).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Serotonin Transport in Human Platelets by Tyrosine Kinase Syk

Pavanetto

Zarpellon

Borgo

et al. 2011

Cell Physiol Biochem

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Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the regulation of numerous neuro-physiological processes. The circulating level of 5-HT is regulated by the membrane transporter SERT present both in the presynaptic nerve terminals and blood platelets. 5-HT transport is a process tightly regulated by a variety of factors including protein phosphorylation. Aim of this study was to ascertain if also the SERT Tyr-phosphorylation mediated by Syk-kinase concurs to the regulation of SERT activity. Indeed we found that 5-HT uptake decreased upon platelet exposure to piceatannol or Syk-inhibitor II, two structurally unrelated inhibitors of the tyrosine-kinase Syk. Tyr-phosphorylation of anti-SERT-immuno-stained proteins in membrane extracts and in anti-SERT-immuno-precipitates, decreased upon platelet treatment with piceatannol, in parallel with a reduction of Syk-activity. Syk was immuno-revealed in the anti-SERT immuno-precipitates, which displayed a piceatannol-sensitive kinase activity towards SERT itself and the Syk-substrate α-sinuclein. Syk inhibitors also caused a decrease of the monensin-induced 5-HT-efflux from platelets and of imipramine binding to them. It is concluded that, in addition to the phosphorylation of SERT mediated by various other kinases, also that catalyzed by Syk might play an important role in the 5-HT transport, likely favoring the transporter conformation exposing the neurotransmitter binding sites.

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“…Since diverse protein regulatory patterns are present in immature megakaryoblasts cells as regards to proplatelets and platelets [49,50], the MEG-01 model should be primarily applied as a developmental model, for investigations regarding molecular differentiation events. For instance, it should be mentioned that a SERT regulatory endoproteolytic cleavage has been observed in human platelets, producing fragments of different size after immunoblot analysis [51,Giannaccini et al,unpublished reports], whereas a single SERT band was resolved in MEG-01 cell extracts, using the same primary antibodies, as here reported. Moreover, megakaryoblastic MEG-01 differentiation is a complex phenomenon leading to up or down-regulation of a variety of genes [52].…”

Section: Discussionmentioning

confidence: 82%

Human Serotonin Transporter Expression During Megakaryocytic Differentiation of MEG-01 Cells

et al. 2009

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The serotonin (5-HT) transporter (SERT) has been found altered in platelets of patients with genetically complex disorders, including mood-anxiety, pain and eating disorders. In this study, we used cell cultures of platelet precursors as models of investigation on mechanisms of SERT regulation: SERT expression was appraised during megakaryocytic differentiation of human megakaryoblastic MEG-01 cells. Cells were cultured for 8 days with 10(-7)M 4-beta-12-tetradecanoylphorbol-13-acetate (beta-TPA) in the presence of 10% fetal bovine serum (FBS) and SERT was assessed by real time PCR, immunofluorescence microscopy, Western blot and [(3)H]5-HT re-uptake. Results revealed that SERT is present in control-untreated MEG-01 cells. beta-TPA-differentiating MEG-01 cells showed a redistribution of SERT fluorescence, diffuse to cell bodies and blebs along with a 3-fold SERT mRNA increase and a moderate raise in SERT protein (1.5/1.4-fold) by immunoblot and re-uptake assays. In summary, we have shown herein that control megakaryoblasts express the SERT protein. SERT is modulated by differentiation events, implying that SERT density in platelets is under the control of megakaryocytopoiesis stages. Differentiation of MEG-01 cells can provide considerable insight into interactions between SERT genetics, transmitter-hormonal/homeostatic mechanisms and signaling pathways.

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Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: Evidence that transporter undergoes endoproteolytic cleavage

Cited by 25 publications

References 47 publications

Serotonin (5-HT) Transport in Human Platelets is Modulated by Src-Catalysed Tyr-Phosphorylation of the Plasma Membrane Transporter SERT

Serotonin (5-HT) Transport in Human Platelets is Modulated by Src-Catalysed Tyr-Phosphorylation of the Plasma Membrane Transporter SERT

Regulation of Serotonin Transport in Human Platelets by Tyrosine Kinase Syk

Human Serotonin Transporter Expression During Megakaryocytic Differentiation of MEG-01 Cells

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