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Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase - PubMed

  • ️Sat Jan 01 2005

Comparative Study

. 2005 Aug 9;102(32):11545-50.

doi: 10.1073/pnas.0501432102. Epub 2005 Jul 29.

Affiliations

Comparative Study

Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase

Harish C Prasad et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.

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Figures

Fig. 1.
Fig. 1.

Location and 5-HT transport activity of human SERT coding variants. (A) Variants are overlayed on a 12 TM model of a single SERT subunit, with NH2 and COOH termini oriented inside the cell. Variants in extramembrane domains are shaded black whereas those in membrane domains are shaded white. (B) 5-HT transport activity of SERT-coding variants in transfected HeLa cells. Data reflect mean values ± SEM of three separate experiments. Means were compared with hSERT cDNA by using a one-way ANOVA followed by Dunnett's test of individual means against hSERT values (*, P < 0.05 taken as significant).

Fig. 2.
Fig. 2.

Analysis of protein expression of hSERT and coding variants. (A) Immunoblots of total cell extracts prepared from HeLa cells transfected with hSERT or one of the variants described in the study. (B) Cell surface expression alterations in hSERT Pro339Leu and Ile425Val. Variants were transfected in parallel with hSERT into HeLa cells, and cell surface transporters were identified by immunoblotting of biotinylated samples, captured as described in Materials and Methods.(C) Quantitative estimations of relative surface density of hSERT, Pro339Leu, and Ile425Val based on densitometry of biotinylation immunoblots. Data reflect mean values of three separate experiments ± SEM. Means were compared with a one-way ANOVA followed by Dunnett's test to compare variant surface expression to that achieved with hSERT (*, P < 0.05 taken as significant).

Fig. 3.
Fig. 3.

Impact of 8BrcGMP and p38 MAPK on hSERT activity. (A) Activity modulation. HeLa cells transfected with hSERT- or hSERT-coding variants were examined for 5-HT transport activities as described in Materials and Methods after pretreatments of cells with either 100 μM 8BrcGMP ± H8 or vehicle for 1 h. (B) Altered p38 MAPK-dependent regulation of hSERT in transfected HeLa cells. Cells transfected with hSERT or hSERT-coding variants were examined after pretreatments of cells with either 1 μM anisomycin ± SB203580 or vehicle for 10 min. Results reflect mean values ± SEM of three separate experiments normalized to each mutant's control measured under vehicletreated conditions (100%). Results in A and B reflect mean values ± SEM of three separate experiments normalized to each mutant's level under vehicletreated conditions (100%). Data were analyzed by a one-way ANOVA with post hoc Bonferonni tests comparing variant to hSERT 8BrcGMP/anisomycin responses with P < 0.05 taken as significant.

Fig. 4.
Fig. 4.

Impact of 8BrcGMP on hSERT surface binding. HeLa cells transfected with hSERT- or hSERT-coding variants were treated with either 100 μM 8BrcGMP ± H8 or vehicle for 1 h. Cells were subjected to cell surface [125I]RTI-55 (5 nM) binding with 5-HT (100 μM) as displacer. Data were analyzed by a one-way ANOVA with post hoc Bonferonni tests comparing variant to hSERT anisomycin responses, with P < 0.05 taken as significant.

Fig. 5.
Fig. 5.

Altered PKG/p38 MAPK sensitivity of 56Ala is evident in native lymphocytes and may involve altered transporter phosphorylation. (A) Lymphocytes were genotyped and cultured as described in Materials and Methods and assessed for 5-HT uptake regulation as described for transfected HeLa cells. Data presented derive from individual lymphocyte lines of determined genotype. Findings were replicated in a separate set of genotyped lines with equivalent results. Uptake levels for each genotype with vehicle-treated conditions were taken as 100%. Transport activities were analyzed by a two-way ANOVA with post hoc Bonferroni tests, with P < 0.05 taken as significant. (B) hSERT Gly56Ala variant displays altered basal phosphorylation and sensitivity to 8BrcGMP. SERTs expressed in transfected HeLa cells were examined 36 h after transfection. (Upper) Representative total extract immunoblot and autoradiogram from SERT immunoprecipitations. (Lower) Quantitation of SERT labeling from phosphorylation studies (n = 3). Values are expressed as mean ± SEM. *, P < 0.01 versus WT-vehicle; ##, P < 0.05 versus WT-vehicle by one-way ANOVA with Bonferroni post hoc analysis.

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References

    1. Jacobs, B. & Azmitia, E. C. (1992) Physiol. Rev. 72, 165-229. - PubMed
    1. Fozzard, J. E. (1989) Peripheral Actions of 5-Hydroxytryptamine (Oxford Univ. Press, New York).
    1. Insel, T. R., Zohar, J., Benkelfat, C. & Murphy, D. L. (1990) Ann. N.Y. Acad. Sci., 574-586. - PubMed
    1. Meltzer, H. Y. (1990) Ann. N.Y. Acad. Sci., 486-499. - PubMed
    1. Gershon, M. D. (1999) Aliment. Pharmacol. Ther. 13, Suppl. 2, 15-30. - PubMed

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