Insulin Receptor-Associated Roles of Serotonin Transporter

Conversely, SERT was described as a regulator of glucose homeostasis. In exploring this feature, the phosphorylation of insulin receptor (IR) was studied in SERT gene knockout (KO) mice. The IR phosphorylation was significantly downregulated only in the placenta, but not in the platelets of SERT-KO mice. In exploring the connection between SERT and the phosphorylation of IR further, studies found a novel interaction between SERT and IR. It appears that SERT-IR association on plasma membrane facilitates the insulin signalling that protects cells against apoptosis. Therefore, this review collated all the known and reported findings and drew a molecular link between SERT and insulin receptor. Insulin Signalling and SERT


Introduction
Insulin signalling plays a major role in maturation and the translocation of serotonin transporter protein (SERT) to the plasma membrane through acting on endoplasmic reticulum (ER) proteins, ERp44. In supporting these reports, SERT was found as arrested at intracellular compartments in diabetes associated placental cells.
Conversely, SERT was described as a regulator of glucose homeostasis. In exploring this feature, the phosphorylation of insulin receptor (IR) was studied in SERT gene knockout (KO) mice.
The IR phosphorylation was significantly downregulated only in the placenta, but not in the platelets of SERT-KO mice. In exploring the connection between SERT and the phosphorylation of IR further, studies found a novel interaction between SERT and IR. It appears that SERT-IR association on plasma membrane facilitates the insulin signalling that protects cells against apoptosis. Therefore, this review collated all the known and reported findings and drew a molecular link between SERT and insulin receptor.

Insulin Signalling and SERT
A well-defined function of serotonin transporter (SERT; SLC6A4) is to regulate the concentration and duration of serotonin (5-HT) at extracellular compartments via a saturable re-uptake mechanism [1]. However, the transport role of SERT is only one of the other functions; SERT has been proposed as a multifunctional plasma membrane protein as protecting cells against apoptosis [2] and a regulator of glucose homeostasis [3,4].
The functions of SERT depends on its correctly folding and cellular location which are regulated by post-translational modifications, such as glycosylation [5][6][7][8], disulfide bond formation [9,10] and oligomerizations [11]. These modifications provide SERT molecules a proper folding, structure, membrane trafficking, association with other proteins and assembly on the plasma membrane. Following these modifications SERT monomers are associated in an oligomeric form [11]. In short, SERT is an oligomeric glycoprotein and insulin signalling plays a major role in the maturation, correctly folding and trafficking to the plasma membrane of SERT [10,12].
Conformational maturation of membrane proteins involves different pathways for post-translational modifications. Formation of intra or intermolecular disulfide bonds is one of the major rate limiting factors. SERT has three cysteine (Cys) residues on external loop. Disulfide bond formation between two residues, C200 and C209, is required for SERT folding, surface expression, and transport activity [9]. Disruption of the disulfide bond by a single mutation of C200 or C209 produces mutants with a terminally exposed thiol that are retained intracellularly [9,10]. However, mutation of both Cys in the pair allows SERT to reach the plasma membrane but compromises transport activity [13]. Previous studies highlighting the importance of a disulfide bond for SERT folding and surface expression suggest a thiol-dependent quality control mechanism in SERT maturation [9,13]. Moreover, the modification of exposed thiols with-mercaptoethanol (MSH) altered the density of SERT molecules on the plasma membrane and the 5-HT uptake rates [10]. Pre-treatment of the cells with MSH led to much lower 5-HT uptake rates when compared to control cells even though MSHtreatment released more SERT molecules to the plasma membrane.

The roles of two endoplasmic reticulum chaperones, ERp44
and ER Oxidase1-(Ero1-L) are well defined in the maturation and quality control of disulfide containing oligomeric proteins [14,15,[16][17][18][19][20][21][22]. These two thioredoxin family members, ER chaperones first bind to the exposed thiol on the protein and form mixed disulfide bonds [23][24][25][26][27][28]. During this process, ERp44 preferentially associates with unassembled subunits of disulfide containing oligomeric proteins [14,19,20]. Various biochemical and molecular biological techniques assess an association between ERp44 and SERT, the role of Ero1-L in the maturation process, and the corresponding impacts on SERT localization and function at the plasma membrane. ERp44 silenced cells led to increased levels of SERT at the cell surface. The functional state of SERT was also compromised based on changes in the mechanism and maximal rate of 5-HT uptake by these cells. Moreover, SERT mutants (C200S, C209S and C109A) with compromised disulfide bond formation and hence structure and function were shown to preferentially associate with ERp44. These studies suggest the importance of disulfide bond formation as a critical step in SERT folding to a fully active form. Also, demonstrated that SERT utilizes ERp44 and Ero1-L, an oxidoreductase in disulfide bond formation process [14,19,20].
These findings from in vitro study models were evaluated in clinical and preclinical study models [23]. In placentas from the diabetes-associated pregnancy, SERT was located with ER [23,24].
In nondiabetic placenta, SERT was dissociated from ERp44 only after insulin treatment which allows the transporter proteins to be translocated to the plasma membrane [23]. However, in diabetic condition, under the lack of insulin signalling ERp44 was associated with Cys200/Cys209 on SERT [23,24]. Consequently, in placentas from the diabetes-associated pregnancy, the glycolytic enzymes could not modify the N-glycosylation sites, Asp208 which is buried between the occupied Cys200 and Cys209 on SERT. Therefore, through two negative feedback mechanisms ERp44 links to SERT in the placentas of diabetes-associated pregnancy. Based on these findings, it was proposed that post-translational modifications of SERT in placental cells are impaired due to the lack of insulin signalling in diabetes-associated pregnancy [23]. These studies demonstrated that insulin signalling is the major mediator in glycosylation, disulfide bond formation and oligomerization of SERT. In diabetes, due to defective insulin signalling, SERT cannot fold properly in the steps of the post-translational modifications.

SERT, A Regulator of Glucose Homeostasis
Upon binding to insulin, the insulin receptor (IR) is phosphorylated and instantly activates intracellular signalling and under diabetic conditions the phosphorylation of IR is impaired.
Insulin signalling is balanced between the phosphorylation and dephosphorylation of IR by tyrosine kinases and phosphatases.
A non-responsive IR to insulin signalling is the most common metabolic complication of obesity and non-insulin-dependent diabetes mellitus, such as type 2 diabetes and gestational diabetes mellitus (GDM), the most common metabolic complication of pregnancy, affecting up to 10-15% of all pregnancies [25,26].
In addition to self-association between -and -subunits, IR also associates with other proteins, which stabilize IR at the cell surface and alter the structure of the receptor [27,28]. Interestingly a physical association between IR molecules and SERT was reported in placental cells, but not in placentas of diabetic subjects [24].
Furthermore, studies with preclinical models report that mice lacking the gene for SERT (SERT-KO) [29][30][31][32] exhibit insulin resistance and glucose intolerance, and progressively develop obesity and hepatic steatosis [33]. These studies clearly throw additional role to SERT in placental cells more than functioning as a transporter, protecting the trophoblast cells against continuous 5-HT signalling.
Based on these and the follow up studies, SERT was determined as regulator of the body fat storage and glucose homeostasis [3,4].
However, this association was altered by insulin treatment as well as by diabetic conditions. The association between SERT-IR was reported as a requirement for the phosphorylation of IR and in turn for the insulin signalling. In SERT gene silenced, SERT-KO mouse placenta the phosphorylation of IR was found significantly reduced (). These findings present a significant role for SERT-IR association in insulin signalling via facilitating the phosphorylation of IR in placental cells.

SERT Against in Cell Death
SERT, as a transporter plays important roles in health and diseases via regulating the levels of 5-HT in extracellular surface. However, SERT has other roles as a plasma membrane protein.
SERT binds IR and facilitates its phosphorylation and regulates the insulin signalling. Further studies explored additional role of SERT in cell death and apoptosis [2].
The histochemical analysis of placentas dissected from 18d pregnant mice lacking the gene encoding SERT showed an abnormal thick band of fibrosis and necrosis [2]. Interestingly, the necrotic insult was primarily located at the junctional zone of the placenta. DNA fragmentation and apoptosis [34]. The findings from these previous studies indicate that SERT on the cell surface was required to prevent programmed cell death. The TUNEL assays located the highest rate of cell death at the maternal side of the placentas in both transgenic mice, but predominantly in the SERT-KO model.
This finding is similar to the necrosis described in human placentas complicated by maternal hypertension [35].
In SERT-KO plasma insulin level is fourfold higher and the glucose level is slightly higher than in WT mice. Therefore, the elevated plasma 5-HT in SERT-KO mice is the likely cause of their resistance to elevated plasma insulin. SERT-KO mice develop obesity and cardiovascular complications, and their embryos show various developmental defects [36]. In collating the reported studies with SERT the review draws a molecular link between SERT and IR in regulating insulin signalling and associated cell death.