GLUTAMATE TRANSPORTERS IN PANCREAS AND EPITHELIA: PHYSIOLOGICAL ROLES AND DYNAMIC REGULATION

Glutamate is the main excitatory neurotransmitter of the mammalian nervous system and is involved in neuronal plasticity, memory and learning. Emerging evidences suggest that glutamate is also present in peripheral tissues, where it plays a role in both cellular homeostasis and in autocrine/paracrine communication as an extracellular signalling molecule [Hediger and Welbourne, 1999; Nedergaard et al., 2002]. The extracellular glutamate concentration is tightly controlled by high affinity glutamate transporters, whose expression and modulation in the peripheral tissues have been poorly investigated. In this work we analyse the high affinity glutamate transporters EAAC1 and GLT1 in epithelia and endocrine pancreas, respectively. EAAC1 was cloned from rabbit intestine [Kanai and Hediger, 1992] and its expression and function have been well characterised in absorptive epithelia, such as intestine and kidney, where it represents the major transporter for the dicarboxylic amino acids [Peghini et al., 1997]. Less is known about the molecular mechanisms that regulate its surface expression and activity. During the past few years, it has become clear that the activity of these transporters can be rapidly regulated by redistribution of proteins to and from the plasma membrane: a process that can be controlled by dynamic protein-protein interactions. Therefore, the research presented in the chapter II focuses on the molecular mechanisms which regulate EAAC1 trafficking in epithelial cells. EAAC1 has a conserved sequence present in the C-terminal domain of EAAC1, that mediates interactions with class I PDZ proteins. In the past years, we demonstrated that the PDZ-target sequence and PDZ proteins are responsible for the retention and stability of EAAC1 at the plasma membrane [D’Amico et al., 2010]. The aim of the present work is to verify whether this PDZ-target sequence is also important for the transporter’s biosynthetic delivery. Our data indicate that PDZ interactions occur early in the biosynthetic pathways and are involved in the ER-to-Golgi trafficking, as well as in Post-Golgi trafficking of EAAC1. Removal of the PDZ motif delays rather than prevents the ER export and the plasma membrane delivery of the transporter, thus indicating that PDZ interactions facilitate the ER-Golgi trafficking. Possibly, PDZ-interactions favour the transporters homo-oligomerization, a process required for the efficient ER export of EAAC1. Alternatively, PDZ domain-proteins may couple EAAC1 with protein complexes required for the efficient fusion of carrier vesicles to the appropriate target membranes. Further studies will be needed to identify the PDZ protein/s involved in the EAAC1 biosynthetic delivery. On the other hand, the presence of glutamate as an intercellular signal mediator in endocrine pancreas is well established [Moriyama and Hayashi, 2003]. In the Central Nervous System (CNS), glutamate may cause cell death by excitotoxicity, that is physiologically prevented by glutamate clearance systems [Choi et al., 1988]. The effect of glutamate on islet viability, the expression of glutamate transporters and their physiological roles in the endocrine pancreas are still unclear. Thus, in the III chapter we examine the effects of glutamate and the function of sodium dependent glutamate transporters in clonal beta-cells and in human isolated islets of Langerhans. We demonstrate that exposure to elevated glutamate concentrations induces a significant cytotoxic effect in pancreatic beta-cells, due to the prolonged activation of ionotropic glutamate receptors. We provide evidence that the key regulator of the extracellular glutamate clearance in the islet is the glial glutamate transporter 1 (GLT1/EAAT2). GLT1 is the only high affinity glutamate transporter expressed in the islets and localizes to the beta-cell plasma membrane. Finally, as diabetes is characterized by selective beta-cell death, and our data indicate that GLT1 controls beta-cell survival, we investigate the expression of GLT1 in type 2 diabetes mellitus (T2DM) patients. We show an altered GLT1 localisation in pancreases from T2DM patients, suggesting a decreased glutamate clearance ability in these subjects. In chapter IV, is reported the experience at University of Texas Health Science Center at San Antonio (UTHSCSA), USA. The aim of this project is to find interactors of IAPP, a protein involved in diabetes, by means of Yeast Two Hybrid Screening, a technique which allows the identification of direct protein-protein interactions. In prospect, this technique will be useful to find proteins that are associated with glutamate transporters and that potentially regulate their expression and function.