The Journal of Neuroscience, October 26, 2005, ():
7 Neuronal Nicotinic Acetylcholine Receptors Are Negatively Regulated by Tyrosine Phosphorylation and Src-Family Kinases
J. Neurosci. Charpantier et al.
25: 9836
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. The ?7 intracellular loop contains two putative sites for tyrosine-phosphorylation. (a) Prediction of phosphorylation sites in human ?7 loop between transmembrane domains TM3 and TM4 by netphos software. Tyr-442 is a consensus phosphorylation site. (b) Sequence comparison of human ?7 (HS) near Tyr-386 with the known tyrosine phosphorylation sites of nAChR subunits from Torpedo californica (TC). Tyr-386 also appears as consensus phosphorylation site. (c) Protein sequence corresponding to the intracellular loop of human ?7. The start methionine is residue 1. Predicted phosphorylation sites are indicated with a diamond, black dot and star for serine, threonine and tyrosine residues, respectively. Arrowheads indicate tyrosine residues, which are replaced by alanine in the ?7 2Y-A receptors. (d and e) Comparison of the sequences surrounding Tyr-386 and Tyr-442 in ?7 among different species reveals a high degree of conservation. Tyr-386 corresponds to a consensus binding motif, NXXY, for phosphotyrosine-binding (PTB) domains, further implying phosphorylation of this residue.
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Supplementary Figure 2. Model of tyrosine kinase and phosphatase pathways that regulate ?7 nAChRs. Genistein and its inactive analog (Igen) are able to act directly on the receptor, but these effects are small. In acting indirectly on a longer time-scale, genistein inhibits members of the Src kinase family, resulting in dephosphorylation of the ?7 receptor, which produces current enhancement. PP2 and SU6656 act similarly, but we did not observe indirect effects. Pervanadate blocks the activity of tyrosine phosphatases, enhancing the phosphorylation of the ?7 receptor resulting in a decrease of the ACh evoked current. Thus tyrosine phosphorylation of ?7 negatively regulates ACh-induced current. As the ?7 receptor is formed by 5 homologous subunits and each subunit has 2 putative phosphorylation sites, it contains up to 10 phosphorylation sites. The role of the binding of SFKs to the loop in ?7 as such is unclear, but it may be a prerequisite for SFK-mediated ?7 phosphorylation.
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Supplementary Table. Effect of kinase and phosphatase inhibitors on the EC50 of ACh for ?7 wild type and ?7 2Y-A mutant receptors. EC50 values are given in µM, nH represents Hill coefficient and n the number of cells tested. Controls were carried out in absence of inhibitors. Co-application indicates that genistein was applied at the same time as ACh, without pre-incubation. In all other treatments, inhibitors were pre-applied: 1-2 h for genistein and its inactive analog (Igen), and 0.5-1 h for pervanadate. (a) Results for wild type ?7. Small but non-significant shifts were observed for genistein and Igen pre-application. (b) Data for mutant (?7 2Y-A) receptor. No significant shift of the EC50 was observed. In control conditions, ACh EC50 is slightly, but not significantly, lower than for wild type ?7.
