Phosphorylation of Threonine Residue 276 Is Required for Acute Regulation of Serotonin Transporter by Cyclic GMP

Sammanda Ramamoorthy,Devadoss J Samuvel,Eric R Buck,Gary Rudnick,Lankupalle D Jayanthi

doi:10.1074/jbc.m611353200

Abstract

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The V(max) value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. (32)P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.

Highlights

5-HT by re-uptake of released 5-HT into presynaptic neurons [1, 2]
The time course of stimulation by 250 ␮M 8-Br-cyclic GMP (cGMP) peaked at 10 –15 min and decreased (Fig. 1A). 5-HT uptake increased maximally (25%) at 250 ␮M 8-Br-cGMP and decreased above that concentration (Fig. 1B). 8-Br-cGMPstimulated 5-HT uptake was blocked almost completely when synaptosomes were incubated with protein kinase G (PKG)-specific inhibitors, KT5823 and (Rp)-8-pcPT-cGMPS, for 30 min prior to exposure to 8-Br-cGMP (Fig. 2)
We examined the molecular mechanisms responsible for the activation of serotonin transporter (SERT) by cGMP using both endogenous SERT in midbrain synaptosomes and heterologously expressed hSERT in CHO-1 cells

Summary

Introduction

5-HT by re-uptake of released 5-HT into presynaptic neurons [1, 2]. the activity of SERT in the presynaptic plasma membrane is a critical factor that determines the strength of serotonergic neurotransmission. We demonstrate that 8-Br-cGMP stimulates 5-HT uptake via PKG activation without altering SERT surface density. Consistent with the stimulatory effect of 8-Br-cGMP on 5-HT uptake in synaptosomes (Fig. 1), exposure of CHO-1 cells expressing wild-type hSERT to 8-Br-cGMP (250 ␮M) for 15 min

Results

Conclusion