Our goal is to define by molecular cloning facilitated diffusion glucose transport proteins in the plasma membrane of several rat cells - adipocytes, striated muscle, hepatocytes, kidney, beta- cells of pancreatic islets, and intestine. Our molecular studies have shown that facilitated diffusion glucose transport proteins comprise a multi-gene family with 4-6 members; we have cloned, sequenced, and characterized two such cDNAs. We will attempt to clone all members of this multigene family by probing lambda gt11 expression cDNA libraries and genomic libraries by hybridization at several stringencies with existing clones, and by use of antibody probes. After cloning and sequencing, we will characterize the physiological importance of these putative transport proteins. Each cDNA will be functionally expressed in a suitable cell, initially in Escherichia coli and murine 3T3 fibroblasts. We will characterize the transport activity of the cloned proteins with respect to sugar specificity, KM ad pharmacological sensitivity. We want to determine the exact type of cell in the issue, and the type of membrane within the cell, that contains each of these transport proteins. We shall generate polyclonal antisera to chemically synthesized peptides that correspond to unique segments of the various glucose transporters. These antisera will be used to detect the presence of the corresponding protein by immunolight- and electron- microscopy and by Western blotting. Subcellular fractionation of adipocytes will also be employed to resolve plasma membrane from microsomes, as will biochemical assays to detect transporters on the cell surface. We want to determine the subcellular localization of all transporters in adipose and muscle cells, any translocation that might be induced by insulin, and also covalent modification of all transporters expressed in these tissues. We will also determine: the identity of the transporter(s) in the membrane of the islet cell that might allow the cells to respond to sense changes in extracellular glucose; and the transporter(s) in the hepatocyte membrane of the islet cell that might allow the cells to respond to sense changes in extracellular glucose;and the transporter(s)in the hepatocyte membrane that transports glucose into or oust of the cell. A long term aim is to understand defects in diabetes relating to sensing of glucose levels by beta cells, and to insulin-stimulated glucose uptake in adipocytes and muscle.
