Abstract

Understanding the physiology of control of glycemia in humans requires detailed knowledge of the mechanisms involved in cellular acquisition of glucose. Because the plasma cell membrane is impermeable to glucose, the participation of glucose carriers to facilitate its uptake from the circulation into cells is required. Fourteen facilitative glucose transporters have been described, collectively known as GLUT transporters. Intensive investigations have revealed the active participation of some of them in glucose homeostasis. GLUT4 is the insulin-dependent glucose transporter expressed in skeletal muscle and adipose tissue; it is normally sequestered in vesicles within these cells and therefore is unable to activate glucose transport until a signal from insulin induces their migration to the cell membrane, where it promotes the uptake of glucose. GLUT2 is the major glucose transporter into pancreatic ?-cells and liver cells and their role is central to the glucose-sensing mechanism that mediates the release of insulin and food intake. GLUT1 is ubiquitously expressed, does not require the action of insulin and facilitates basal glucose uptake in all cells; it therefore plays an important role in the nervous system, which relies almost exclusively on glucose to meet its energy demands. Just two genes of the SLC2 family have been linked to genetic diseases in humans: the GLUT1 deficiency syndrome (GLUT1DS) and the Fanconi-Bickel syndrome, which arise from inheritable mutations on the SLC2A1 and SLC2A2 genes encoding GLUT1 and GLUT2, respectively. Our proposal is centered to identify structural regions and specific amino acid residues of functional and regulatory importance into human GLUT1 and GLUT2. The central aim of this proposal is to elucidate the mechanism of transport and regulation of the glucose transporter GLUT1 and GLUT2. Specifically, by a detailed functional analysis of site-directed mutant using different substrates and inhibitors under several assays settings, and chemical modification followed by mass spectrometry mapping, we will collect functional and structural data for GLUT2 and GLUT1 glucose transporters. These approach will permits us to delineate the molecular mechanism of substrate migration through the transport channel, identify the substrate binding site, recognize the regulatory domains involved in the binding of inhibitors, detect the structural regions important for transporter function and regulation and to probe new candidate regulators. Our hypotheses establish that besides its substrate site GLUT1 harbors regulatory sites able to bind different blockers and that the structural information collected will permits us to identify amino acid residues defining these allosteric sites. We also propose that the analysis of some GLUT2 pathogenic mutants will allow us to define the functional and structural characteristics of this transporter. We assume that the structure of GLUT2 is similar to that of GLUT1, so the information gathered for GLUT1 will allow us to identify amino acid residues and structural domains of functional and regulatory importance for the GLUT2 transporter. The specific aims of this proposal are: 1) Identification and functional characterization of regulatory domains on GLUT1. 2) Identification and functional characterization in GLUT2 of amino acid residues conforming substrate binding sites glucose and the translocation channel for glucose. 3) Determination of GLUT2 topology by accessibility to covalent modification detected by mass spectrometry mapping. The long term goal of this project is to obtain basic knowledge on the structure-function relationship in the GLUT1 and GLUT2 transporters to develop procedures to modify the cellular uptake of glucose with potential biomedical applications. Thus, our main efforts are focused to identify specific amino acids residues or regions of functional and regulatory importance on these carrier proteins. We expect to generate detailed structural and functional information that will define the relationship between the molecular architecture of GLUT1 and of GLUT2 and the mechanism and regulation of glucose transport and sensing in humans. We look forward that the information obtained about the fine architecture of these regulatory domains, will allow the design of new regulatory ligands for these transporters or to devise new strategies for therapeutic interventions. We can imagine several scenarios in which this information can be very beneficial. For instance, malignant cells have high rates of anaerobic glycolysis and increased requirements for ATP production, and RNA and protein analysis has suggested an up-regulation of GLUT1 expression in different human cancer tissues, while GLUT2 is overexpressed in hepatoma cells. A disruption of glucose uptake via glucose transporter proteins may then alter the metabolism of malignant cells, leading to reduced tumor growth.