The basic amino acid transporter (system y+) is the primary mechanism for cellular uptake of arginine and lysine; it is the first mammalian amino acid transporter to be cloned, and the first to be expressed and studied by electrophysiological methods in Xenopus oocytes. The y+ molecule also functions as the receptor that mediates attachment and infection of ecotropic host-range murine retroviruses that produce leukemia.
The specific aims of this proposal involve structure-function studies on the y+ molecule and isolation and characterization of genes related to it.
Aim 1 is to express y+ in oocytes and to determine its transport functions using voltage clamp methods, both with microelectrodes and with the 'cut- open' oocyte technique. A biophysical of model time-, voltage-, and concentration-dependence of substrate uptake and efflux will be developed.
Aim 2 is to clone and express genes encoding other transporters. Using a partial-length cDNA encoding the y+-related gene Tea, the full length cDNA will be cloned and expressed, and its transport functions characterized. Other genes homologous to y+ will be identified by screening cDNA libraries and similarly tested. Another strategy for isolating amino acid transporter genes will utilize an oocyte expression cloning scheme to screen for expression of radiolabeled substrate uptake.
Aim 3 involves structure-function studies on the system y+ protein; comparisons with related transporters will allow chimera construction and site-directed mutagenesis to map functional domains in the y+ molecule.
In Aim 4 studies will be done to analyze the structural features of y+ important for its role in infection and in the natural resistance of non-murine species. Site-directed mutants and chimeric molecules made in aim 3 will be expressed in mammalian cells and analyzed for abilities to bind virus and to mediate infection. The results will be of significance in cell biology because they provide information about a critical class of transporter molecules, in virology because they will suggest how retroviruses invade cells, and in medicine because they promise to provide new understanding of common inherited diseases that result from abnormal amino acid transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048709-03
Application #
2186228
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1993-08-01
Project End
1997-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Type
Organized Research Units
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Kavanaugh, M P; Bendahan, A; Zerangue, N et al. (1997) Mutation of an amino acid residue influencing potassium coupling in the glutamate transporter GLT-1 induces obligate exchange. J Biol Chem 272:1703-8
Seyfang, A; Kavanaugh, M P; Landfear, S M (1997) Aspartate 19 and glutamate 121 are critical for transport function of the myo-inositol/H+ symporter from Leishmania donovani. J Biol Chem 272:24210-5
Sonders, M S; Zhu, S J; Zahniser, N R et al. (1997) Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants. J Neurosci 17:960-74
Bismuth, Y; Kavanaugh, M P; Kanner, B I (1997) Tyrosine 140 of the gamma-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition. J Biol Chem 272:16096-102
Zerangue, N; Kavanaugh, M P (1996) Interaction of L-cysteine with a human excitatory amino acid transporter. J Physiol 493 ( Pt 2):419-23
Wang, H; Klamo, E; Kuhmann, S E et al. (1996) Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter. J Virol 70:6884-91
Zerangue, N; Kavanaugh, M P (1996) ASCT-1 is a neutral amino acid exchanger with chloride channel activity. J Biol Chem 271:27991-4
Kavanaugh, M P; Kabat, D (1996) Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family. Kidney Int 49:959-63
Wadiche, J I; Amara, S G; Kavanaugh, M P (1995) Ion fluxes associated with excitatory amino acid transport. Neuron 15:721-8
Drew, M E; Langford, C K; Klamo, E M et al. (1995) Functional expression of a myo-inositol/H+ symporter from Leishmania donovani. Mol Cell Biol 15:5508-15

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