Serotonin (5HT) is a vasoconstrictor that also acts as a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5HT-signaling causes maternal and prenatal morbidity and mortality via mediating high blood pressure and neuroanatomical abnormalities, respectively. As a mitogen, the initial action of 5HT is important for the development of the embryo. However, the embryo does not synthesis 5HT but receives it from the maternal-embryonic circulation via a specific 5HT transporter (SERT) on the membranes of the trophoblast cells. Therefore, regulation of the transporter's activity constitutes a key mechanism for the flow of 5HT from mother to the developing embryo. SERT assembles into a number of higher-order complexes and functions as an oligomer and not simply as individual monomers in an oligomeric complex. SERT functions most favorably in the oligomeric form. However, the mediators that facilitate the oligomerization of SERT proteins have not been identified yet. The only study of this subject shows that glucose, at diabetes-like concentrations, down-regulates 5HT uptake rates of placental SERT through inhibiting its oligomerization. In human placental trophoblastoma (JAR) cells, our preliminary assays showed that SERT proteins colocalized with and bound to ERp44, endoplasmic reticulum (ER) located chaperone. ERp44 plays a critical role in the release of proteins from ER via binding to Ero1-L1. Our next preliminary studies showed that glucose, at diabetes-like concentrations, down regulates the mRNA level expression of ERp44. Therefore, these findings strengthen our confidence on the involvement of ERp44 in oligomerization of SERT proteins. Further, we showed that two mutant forms of SERT could neither associate with each others nor with ERp44. Based on our studies, we hypothesize that ERp44 has a defined role in the assembly of SERT proteins by forming disulfide bridges between SERT monomers. Once the oligomerization of SERT is completed, transporter in the matured form dissociate from the ERp44-Ero1-L1 complex and leave ER to enter the membrane trafficking. The proposed study will utilized pulse-chase experiments and immunoprecipitation assays to monitor the biogenesis of SERT proteins in (i) JAR cells, and in (ii) ERp44 or Ero1-L1 knock-down JAR cells. Also introducing Cys residues at different positions on the second external loop of SERT and then cross-linking them, we will assess the impact of additional intramolecular disulfide bonds on Ero1-L1- ERp44-SERT association. Overall, this project will provide the first detailed information on the involvement of ERp44-Ero1-L1 in the oligomerization of placental SERT proteins and shed light on the mechanism in which glucose prevents the oligomerization of placental SERT. The importance of understanding the structure, function, and regulation of SERT is underscored by the observations that placental SERT may be not functionally active during diabetes, hypertension, stroke, and other cardiovascular diseases during pregnancy.

Public Health Relevance

As a mitogen, the initial action of serotonin (5HT) is important for the development of embryo however embryo does not synthesis 5HT but receives it from the maternal- embryonic circulation via a specific 5HT transporter (SERT) on the membranes of the trophoblast cells. Therefore, regulation of the transporter's activity constitutes a key mechanism for the flow of 5HT from mother to the developing embryo. SERT assembles into a number of higher-order complex and function as oligomers and not simply as individual monomers in an oligomeric complex. SERT functions most favorable in the oligomeric form however, the mediators which facilitates the oligomerization of SERT proteins have not been identified yet. This is the first study that identified two ER chaperones, ERp44 and Ero1-L1, in facilitating the oligomeric expression of SERT in placental cells. The importance of understanding the structure, function, and regulation of placental SERT is underscored by the many clinical and experimental studies which have implicated abnormal or inadequate 5HT accumulation in placenta and placental vascular bed. Our findings will provide data that could facilitate novel strategies aimed at regulating the functional efficiency of placental SERT and subsequently control 5HT-mediated embryonic development.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
5R03HD058697-02
Application #
7807032
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Willinger, Marian
Project Start
2009-05-01
Project End
2011-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
2
Fiscal Year
2010
Total Cost
$70,971
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Hadden, Coedy; Fahmi, Tariq; Cooper, Anthonya et al. (2017) Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway. J Cell Physiol 232:3520-3529
Li, Yicong; Hadden, Coedy; Cooper, Anthonya et al. (2016) Sepsis-induced elevation in plasma serotonin facilitates endothelial hyperpermeability. Sci Rep 6:22747
Joo, Joung Hyuck; Wang, Bo; Frankel, Elisa et al. (2016) The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis. Mol Cell 62:491-506
Li, Yicong; Cooper, Anthonya; Odibo, Imelda N et al. (2016) Discrepancy in Insulin Regulation between Gestational Diabetes Mellitus (GDM) Platelets and Placenta. J Biol Chem 291:9657-65
Basnet, Kristen M; Bentley-Lewis, Rhonda; Wexler, Deborah J et al. (2016) Prevalence of Intervillous Thrombi Is Increased in Placentas from Pregnancies Complicated by Diabetes. Pediatr Dev Pathol 19:502-505
Fraer, Mony; Kilic, Fusun (2015) Serotonin: a different player in hypertension-associated thrombosis. Hypertension 65:942-8
Li, Yicong; Hadden, Coedy; Singh, Preeti et al. (2014) GDM-associated insulin deficiency hinders the dissociation of SERT from ERp44 and down-regulates placental 5-HT uptake. Proc Natl Acad Sci U S A 111:E5697-705
Mercado, Charles P; Quintero, Maritza V; Li, Yicong et al. (2013) A serotonin-induced N-glycan switch regulates platelet aggregation. Sci Rep 3:2795
Mercado, Charles P; Byrum, Stephanie; Beggs, Marjorie L et al. (2013) Impact of elevated plasma serotonin on global gene expression of murine megakaryocytes. PLoS One 8:e72580
Freyaldenhoven, Samuel; Li, Yicong; Kocabas, Arif M et al. (2012) The role of ERp44 in maturation of serotonin transporter protein. J Biol Chem 287:17801-11

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