Serotonin(1A)-receptor-dependent signaling proteins in mouse hippocampus

Abstract

The serotonin(1A) receptor (5-HT(1A) R) knock-out mouse (KO) is a widely used animal model for anxiety and cognitive function and regulation of signaling cascades by this receptor has been reported. We aimed to determine individual representatives of signaling cascades in order to screen 5-HT(1A) R-dependent signaling proteins (SPs). Hippocampal proteins from wild type and 5-HT(1A) R KO mice were extracted, run on two-dimensional gel electrophoresis, proteins were identified by MALDI and nano-ESI-LC-MS/MS and SPs were quantified by specific software. Nucleoside diphosphate kinase A (NDK A, synonym: nm23), Dual specificity mitogen-activated protein kinase kinase 1 (MAPKK1, synonym: MEK), Serine/threonine-protein phosphatase PP1-gamma catalytic subunit (PP-1G), Septin-5, were reduced in the KO mice. Novel phosphorylation sites at T386 on MAPKK1 and at S225 and Y265 on Septin-5 were observed. MAPKK1 and PP-1G are known 5-HT(1A) R-dependent signaling compounds and are in agreement with receptor knock-out and septin-5 is involved in serotonin transport, although regulation by 5-HT(1A) R has not been reported. 5-HT(1A) R - dependent levels for NDK A have not been demonstrated so far and we herewith propose a role for NDK A in 5-HT(1A) R signaling. Reduced SP levels along with findings of two novel phosphorylation sites may be relevant for interpretation of previous and the design of future studies on this receptor system.

Fig. 1

A combined map of proteins from WT and KO mice is given. Spots are identified and UniProtKB accession numbers are provided. Proteins with significantly different levels are shown in “white”.

Fig. 2

Images of individual spots on 2-DE in WT and KO mice.

Fig. 3

Identification of phosphorylated tryptic peptides by nano-ESI-LC/MS/MS. a) MS/MS spectrum of dual specificity mitogen-activated protein kinase kinase 1 (P31938) peptide ³⁶³RSDAEEVDFAGWLCSTIGLNQPSTPTHAASI³⁹³ (m/z = 1137.53, 3+) containing T386 phosphorylation.; b) MS/MS spectrum of Septin-5 (Q9Z2Q6) peptide ²¹³FGIHVYQFPECDSDEDEDFKQQDR²³⁶ (m/z = 1028.87, 3+) containing S225 phosphorylation; c) MS/MS spectrum of Septin-5 (Q9Z2Q6) peptide ²⁶²GRLYPWGIVEVENQAHCDFVK²⁸² (m/z = 866.19, 3+) containing Y265 phosphorylation.

Fig. 4

Western blots showing immunoreactive bands for MAPKK1, NDKA and the two expression forms of PP-1G including results from immunoblotting. A single actin band was comparable between individuals and groups and used as loading control.