Abstract
Arginine transport is important for a number of biological processes in vertebrates, and its transport may be rate-limiting for the production of nitric oxide. The majority of L-Arg transport is mediated by System y+, although several other carriers have been kinetically defined. System y+ cationic amino acid transport is mediated by proteins encoded by a family of genes, Cat1, Cat2, and Cat3. High affinity L-arginine transport was investigated in embryonic fibroblast cells derived from Cat1 knockout mice that lack functional Cat1. Both wild type and knockout cells transport arginine with comparable Km and Vmax. However, the apparent affinity for lysine transport was 2.4 times lower in Cat1(-/-) cells when compared with wild type cells, a property characteristic of Cat3-mediated transport. Northern analysis-documented Cat2 mRNA increased 2-fold, whereas Cat3 mRNA levels increased 11-fold in Cat1(-/-) relative to Cat1(+/+) cells. The low affinity Cat2a mRNA was not detectably expressed in these cells. Even though Cat3 expression is normally limited to adult brain, there was a large increase in the amount of Cat3 protein present at the plasma membrane of Cat1(-/-) embryonic fibroblast cells. These results suggest that Cat3 compensates for the loss of functional Cat1 in cells derived from Cat1 knockout mice and mediates the majority of high affinity arginine transport.
Highlights
Three high affinity cationic amino acid transporters, Cat1, Cat2, and Cat3, were cloned serendipitously in 1989 [10], 1990 [11], and 1997 [12], respectively. Whereas these transporters share only about 61% amino acid sequence identity they were found to be kinetically indistinguishable with very similar Km values for L-arginine in oocytes [13, 14]
Cat11 appears to have a greater capacity than Cat2 for trans-stimulation [14], whereas an alternately spliced transcript of Cat2 encodes a kinetically distinct low affinity form of Cat2a [15]
Taking advantage of the differential affinity for lysine and a cell line established from the Cat1Ϫ/Ϫ embryonic fibroblasts, we investigated arginine and lysine uptake into Cat1Ϫ/Ϫ MEF cells
Summary
Three high affinity cationic amino acid transporters, Cat1, Cat2, and Cat3, were cloned serendipitously in 1989 [10], 1990 [11], and 1997 [12], respectively. 1 The abbreviations used are: Cat, cationic amino acid transporter; MEF, mouse embryonic fibroblast; gapdh, glyceraldehyde-3-phosphate dehydrogenase. The disintegrations per min (dpm) in the cells following washing represented the non-saturable and binding components of arginine uptake into mouse embryonic fibroblasts.
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