Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase.
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.
Prasad
Harish C
HC
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
Zhu
Chong-Bin
CB
McCauley
Jacob L
JL
Samuvel
Devadoss J
DJ
Ramamoorthy
Sammanda
S
Shelton
Richard C
RC
Hewlett
William A
WA
Sutcliffe
James S
JS
Blakely
Randy D
RD
D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D015870:Gene Expression; D014644:Genetic Variation; D006367:HeLa Cells; D004854:Herpesvirus 4, Human; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D012701:Serotonin; D017869:Cyclic GMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases