The catecholamine, norepinephrine (NE) governs a spectrum of physiologic processes from vasoconstriction and heart rate to attention and motivation. NE signaling is dynamically regulated by a diverse set of macromolecules including NE transporters (NETs). NETs expressed in the CNS and periphery are important targets for tricyclic antidepressants and psychostimulants. Altered NE transport is documented in cardiovascular diseases and brain disorders. Second messenger linked kinase mediated regulation of human genetic variants of NET is altered in some cardiovascular phenotypes signifying the search for underlying mechanisms of NE transport regulation. NET contains putative phosphorylation sites for several kinases including protein kinase C (PKC). Recently, we demonstrated that, in rat placental trophoblasts, PKC activation enhances native NET internalization via lipid rafts and stimulates transporter phosphorylation. Following the studies on native NET regulation, we directed our studies to explore the role of NET phosphorylation in NE transport regulation. Using a human placental trophoblast cell line expressing hNET (HTR-hNET cells), we demonstrate that a double mutation at a predicted PKC phosphorylation motif of hNET prevents neurokinin 1 receptor (NK1R)/PKC-mediated transporter regulation and phosphorylation. Excessive neurokinin secretion is linked to pre-eclampsia, and thus, the regulation of placental NET by NK1R activation suggests physiological relevance of such regulation in the maintenance of a normal pregnancy. Based on these observations, we propose to test a specific hypothesis that NET phosphorylation is required for NE transport regulation in two separate Specific Aims.
In Specific Aim I, we will identify the signals, specific sites and motifs involved in NK1R/PKC regulated NET phosphorylation and expression to explore the relationship between NET phosphorylation and NE transport.
Specific Aim II will test the hypothesis that NK1R/PKC regulated NET phosphorylation and interaction with transporter-associated proteins occur in lipid rafts, and that this association establishes transporter distribution and function in an activity-dependent manner to cope with the demands in the milieu of incoming signals. The results from this research will provide information that could be of use in the development of new therapeutic strategies aimed at NE transport regulation in the treatment of both cardiovascular diseases and brain disorders. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM081054-01A2
Application #
7196942
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Dunsmore, Sarah
Project Start
2007-02-01
Project End
2012-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
1
Fiscal Year
2007
Total Cost
$220,460
Indirect Cost
Name
Medical University of South Carolina
Department
Neurosciences
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Mannangatti, Padmanabhan; NarasimhaNaidu, Kamalakkannan; Damaj, Mohamad Imad et al. (2015) A Role for p38 Mitogen-activated Protein Kinase-mediated Threonine 30-dependent Norepinephrine Transporter Regulation in Cocaine Sensitization and Conditioned Place Preference. J Biol Chem 290:10814-27
Arapulisamy, Obulakshmi; Mannangatti, Padmanabhan; Jayanthi, Lankupalle D (2013) Regulated norepinephrine transporter interaction with the neurokinin-1 receptor establishes transporter subcellular localization. J Biol Chem 288:28599-610
Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Arapulisamy, Obulakshmi et al. (2012) Tyrosine phosphorylation of the human serotonin transporter: a role in the transporter stability and function. Mol Pharmacol 81:73-85
Mannangatti, Padmanabhan; Arapulisamy, Obulakshmi; Shippenberg, Toni S et al. (2011) Cocaine up-regulation of the norepinephrine transporter requires threonine 30 phosphorylation by p38 mitogen-activated protein kinase. J Biol Chem 286:20239-50
Boger, H A; Mannangatti, P; Samuvel, D J et al. (2011) Effects of brain-derived neurotrophic factor on dopaminergic function and motor behavior during aging. Genes Brain Behav 10:186-98
DeVito, L M; Balu, D T; Kanter, B R et al. (2011) Serine racemase deletion disrupts memory for order and alters cortical dendritic morphology. Genes Brain Behav 10:210-22
Ramamoorthy, Sammanda; Shippenberg, Toni S; Jayanthi, Lankupalle D (2011) Regulation of monoamine transporters: Role of transporter phosphorylation. Pharmacol Ther 129:220-38
Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Arapulisamy, Obulakshmi et al. (2010) Involvement of threonine 258 and serine 259 motif in amphetamine-induced norepinephrine transporter endocytosis. J Neurochem 115:23-35
Ramamoorthy, Sammanda; Samuvel, Devadoss J; Balasubramaniam, Annamalai et al. (2010) Altered dopamine transporter function and phosphorylation following chronic cocaine self-administration and extinction in rats. Biochem Biophys Res Commun 391:1517-21
Zapata, Agustin; Kivell, Bronwyn; Han, Yang et al. (2007) Regulation of dopamine transporter function and cell surface expression by D3 dopamine receptors. J Biol Chem 282:35842-54

Showing the most recent 10 out of 11 publications