Role of p11 in cellular and behavioral effects of 5-HT4 receptor stimulation

Abstract

p11 (S100A10), a member of a large family of S100 proteins, interacts with serotonin receptor 1B (5-HTR1B), modulates 5-HT1B receptor signal transduction, and is required for antidepressant responses to activation of this receptor. In the current study, we investigated the specificity of the interaction between 5-HTR1B and p11 by screening brain-expressed S100 proteins against serotonin and noradrenergic receptors. The data indicate that p11 is unique among its family members for its interactions with defined serotonin receptors. We identify a novel p11-interacting receptor (5-HTR4) and characterize the interaction between p11 and 5-HTR4, demonstrating that (1) p11 and 5-HTR4 mRNA and protein are coexpressed in brain regions that are relevant for major depression, (2) p11 increases 5-HTR4 surface expression and facilitates 5-HTR4 signaling, and (3) p11 is required for the behavioral antidepressant responses to 5-HTR4 stimulation in vivo. The essential role played by p11 in modulating signaling through 5-HT4 as well as 5-HT1B receptors supports the concept that this protein may be a key determinant of vulnerability to depression.

Figure 1.

p11 is the only S100 protein interacting with serotonin receptors (5-HTR1B, 5-HTR1D, and 5-HTR4). a, Using a yeast-based screening system, full-length S100 proteins were analyzed for interactions with the third intracellular loops of serotonin receptors. Blue boxes indicate positive interactions, darker blue representing stronger interaction. b, c, GST-pulldown assay confirmed the interaction between p11 and 5-HTR4 proteins. GST-5-HT4 or GST-5-HT6 (the entire intracellular loop 3 fused to the GST domain) were expressed in E. coli, purified, and stained with Coomassie Blue in (b). c, In vitro translated full-length p11 (left) or luciferase (right) was incubated with immobilized GST–fusion proteins (GST as negative control and GST-5-HT6 as specificity control). Retained radio-labeled products were analyzed by SDS-PAGE. 2% input used for luciferase pull-down is shown. Size markers are indicated on the left.

Figure 2.

Colocalization of p11 and 5-HTR4 mRNA in depression-related brain regions. mRNAs were detected in wild-type mice by double fluorescent in situ hybridization (FISH) using dig-labeled p11 (red) and fluorescein-labeled 5-HTR4 (green) probes with DAPI (blue) nuclear counterstain. a–f, Low magnification images stained with DAPI illustrate brain regions included in analysis: (a) cortex, (b) striatum, (c) amygdala, (d) dentate gyrus of hippocampus, and (e) CA1 pyramidal cell layer of hippocampus. (f) is an example in which some p11-positive cells do not express 5-HTR4. a'–f' correspond to the field included in the white boxes in a–f. Dotted boxes in a'–f' are magnified to the right. White arrows indicate cells expressing both p11 (red) and 5-HTR4 (green) mRNAs against the DAPI (blue) counterstain. Yellow arrow indicates one 5-HTR4 cell that does not coexpress p11, and open yellow arrows indicate cells expressing p11, but not 5-HTR4.

Figure 3.

p11 protein colocalizes with 5-HTR4. a–e, Immunohistochemistry was performed using antibodies recognizing p11 (red) or GFP (green) in brain sections from BAC transgenic mice overexpressing eGFP under the 5-HTR4 promoter. Confocal micrographs taken from (a) cingulate cortex, (b) striatum, (c) amygdala, (d) dentate gyrus of the hippocampus and (e) CA1 pyramidal cell layer of the hippocampus at 40× magnification. White boxes in a–e are enlarged to the right to illustrate single cells (white arrows) expressing both p11 (red) and GFP/5-HT4 (green). Open arrow in a points to p11-positive cell that does not express GFP.

Figure 4.

p11 increases 5-HTR4 expression at the plasma membrane. a–c, COS7 cells were transfected with (a) p11 (green) and 5-HTR4 (red), (b) 5-HTR4 alone or (c) p11 alone. Regions included in the inset are marked by white dotted boxes. White arrow head in a indicates colocalization of p11 and 5-HTR4 at cell surface. d, Biotinylation experiment from transfected COS7 cells. Data were normalized to the amount of 5-HTR4 present in cells transfected with only 5-HTR4 (left, Western blot; right, quantification). e, 5-HTR4 protein expression in plasma membrane enriched fraction versus total hippocampal lysate from p11 WT or KO mice (left, Western blot; right, quantification). f, In vitro 5-HTR4 ligand binding assay using [³H]GR113808 in cortex, hippocampus, and striatum of p11 KO or WT mice. g, Representative image for 5-HTR4 ligand binding in striatum (arrows) of p11 WT or KO mice (for quantification, see Results). All data are presented as mean ± SEM, *p < 0.05.

Figure 5.

p11 facilitates signaling through 5-HTR4. a, Effect of 5-HT (1 μm) on cAMP levels in COS7 cells transfected with p11, 5-HTR4, p11 and 5-HTR4, 5-HTR6, or p11 and 5-HTR6. b, Effect of stimulation with 5-HTR4 partial agonist RS67333 (10 μm) in primary neuronal cultures derived from p11 WT or KO mice. Increase in cAMP levels in response to RS67333 was blunted by >50% in p11 KO cultures compared with WT. c, Quantification and representative image from Western blotting analysis of transfected COS7 cells in the presence of serotonin (1 μm). Cotransfection of p11 with 5-HTR4 increased phosphorylation of MAPK compared with 5-HTR4 alone (1 = mock, 2 = p11, 3 = 5-HTR4, 4 = 5-HTR4 and p11, 5 = 5-HTR6, and 6 = 5-HTR6 and p11). d, Primary cortical neuronal cultures derived from p11 WT or KO mice stimulated with RS67333. RS67333 increased phosphorylation of MAPK in WT, but not in KO cultures. All data are presented as mean ± SEM, *p < 0.05, **p < 0.01.

Figure 6.

p11 is required for the behavioral antidepressant actions of a selective 5-HTR4 partial agonist (RS67333). p11 knock-outs and wild-type littermate controls were injected with RS67333 or saline vehicle for 3–5 d before behavioral testing. Immobility is an index of antidepressant activity in both the TST and FST. a, c, Consistent with the action of an antidepressant, RS67333 reduced time spent immobile in the TST (a) and FST (c) in wild-type mice but had no effect in p11 knock-out mice. In the TST (a) EMDT, an agonist for 5-HTR6 (a 5-HT receptor that does not interact with p11) reduced immobility in both p11 KO and WT mice. b, Treatment of WT mice with GR125487, a 5-HTR4 antagonist, blocked the antidepressant-like effect of RS67333 in the TST. d, RS67333 had an anxiolytic effect in the Open Field, reducing thigmotaxis in wild-type mice, but not in p11 knock-out mice. All data are presented as means ± SEM *p < 0.05, **p < 0.01