Within the last ten years, significant progress has been made to extend our understanding of facilitated glucose transport, often the rate limiting step to its metabolism. Recognition that transport is mediated by a family of closely related proteins has allowed the study of regulation at a molecular level. The focus of this application is on the regulation of the constitutive transporter, GLUT1. The hypothesis that directs our research is that GLUT1 activity is regulated by a unique protein whose synthesis is dependent on glucose availability. This regulation occurs within the physiological range of circulating glucose, which makes this project important within that context. Thus, our goal is to identify a protein that modulates transport activity in 3T3=-L1 adipocytes, a model system for adipocyte differentiation. The first objective is to demonstrate colocalization of a transport activating protein in GLUT1-containing vesicles from either plasma membranes or intracellular vesicles. We will accomplish this task using antibodies specific for GLUT1 which immunoprecipitate the transporter, along with other proteins contained in the same vesicles as GLUT1. Secondly, we will construct two types of GLUT1 fusion proteins as affinity probes to identify transport binding proteins in 3T3-L1 adipocytes, which will both complement the first objective and potentially provide new information regarding binding properties of the transport activating protein. Sequence information will be used to generate antibodies to characterize the glucose-dependent regulation of the transport activating protein. Together, these studies will define the mechanism by which physiological levels of glucose regulates the activity of the transporter.
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