Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization

Obulakshmi Arapulisamy,Padmanabhan Mannangatti,Lankupalle D Jayanthi

doi:10.1074/jbc.m113.472878

Abstract

Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NET·NK1R complexes. Receptor activation resulted in the internalization of NET·NK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NET·NK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NET·NK1R complexes to raft-rich membrane fractions. Importantly, PKCα was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DM·NK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr(258) + Ser(259) motif-dependent translocation of NET·NK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKCα to coordinate spatially restricted NET regulation.

Highlights

Neurokinin-1 receptor (NK1R) activation down-regulates norepinephrine transporter (NET)
We show that NET/NK1R association occurs on the plasma membrane and the NET1⁄7NK1R complex internalizes following receptor activation
Our recent study showed the expression of NET in rat prefrontal cortex (PFC) and ventral striatum (VST) [29]

Summary

Background

Neurokinin-1 receptor (NK1R) activation down-regulates norepinephrine transporter (NET). SP treatment failed to translocate NET-DM1⁄7NK1R complexes from non-raft fractions to raft fractions These results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr258 ؉ Ser259 motif-dependent translocation of. NK1R is localized in lipid rafts [15, 16] and activation of NK1R stimulates translocation of PKC into lipid rafts, where it interacts with the receptor and causes its desensitization [15]. We show that NET/NK1R association occurs on the plasma membrane and the NET1⁄7NK1R complex internalizes following receptor activation. We show that the previously identified PKC-resistant Thr258/Ser259 motif in NET mediates NET/NK1R translocation to the rafts These results suggest that subcellular localization of NET is established via regulated NET/NK1R interaction and raft translocation. Both NE and NK1 signals are implicated in mental disorders such as depression and attention-deficit hyperactivity disorder (19 –23)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION