Cloning and Characterization of a Brain-specific Cationic Amino Acid Transporter

Abstract

rCAT3 (rat cationic amino acid transporter 3), a cDNA that encodes a novel member of the murine CAT family was isolated. The protein encoded by rCAT3 contained 619 amino acids, 53-58% of which were identical with those of the murine CAT family proteins previously described (mouse CAT1, CAT2a, CAT2b, and rat CAT1). Transient expression of rCAT3 and L-[14 C]arginine incorporation experiments in COS7 cells verified a high affinity system y ؉ transporter activity of rCAT3. First, rCAT3-mediated L-[ 14 C]arginine incorporation was timedependent and saturable with half-saturation constant (K m ) values of 103 ؎ 12 M (mean ؎ S.E., n ‫؍‬ 3). Second, the incorporation was specific for cationic amino acids as evidenced from the inhibition by L-arginine, L-lysine, and L-ornithine. Third, neither sodium nor chloride ions in the extracellular medium were required for the activity. Fourth, the incorporation was inhibited by high potassium-induced membrane depolarization. On Northern blot using RNAs from various rat tissues, the expression of rCAT3 mRNA was restricted to the brain. These results indicated a role of rCAT3 in the system y ؉ transporter activity in the nervous tissue.

Increased Cat3-mediated Cationic Amino Acid Transport Functionally Compensates in Cat1 Knockout Cell Lines

“…However, in our hands we were unable to entirely block the immunofluorescence signal by preincubation with the Cat3-immunizing peptide. Therefore the Cat3 antibody was used to probe for Cat3 expression in mouse brain where Cat3 expression has been described (12,16). Immunopositive regions of the normal adult mouse brain were in agreement with the in situ data previously reported (12) (data not shown).…”

“…Ϫ/Ϫ cells is characteristic of Cat3 transport (12,16). These data indicate that both Cat1 ϩ/ϩ and Cat1 Ϫ/Ϫ MEF cells possess System y ϩ transport activity.…”

“…In both species, Cat3 expression was localized to the brain although it was widely expressed in the 13.5-day postcoital mouse embryo. Unlike Cat1 or Cat2, Cat3 has different K m values for L-arginine (40 -60 M) and L-lysine (115-165 M) uptake in Xenopus oocytes (12) and COS7 cells (L-Arg, 103 Ϯ 12 M; L-Lys, 147 Ϯ 22 M) (16). Hence, Cat3 can be kinetically distinguished from Cat1, Cat2, and Cat2a by their differential affinity to L-Lys.…”

“…Cat1 1 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). Cat3 was cloned from mouse (12) and rat (16). In both species, Cat3 expression was localized to the brain although it was widely expressed in the 13.5-day postcoital mouse embryo.…”

Arginine metabolism: nitric oxide and beyond

“…Recent studies have identified cDNAs encoding two transmembrane proteins, CAT-1 and CAT-2(B), which have amino acid transport properties consistent with system y + [165,167]. A cDNA encoding rCAT3, a brain-specific protein which also exhibits system-y + activity, has recently been isolated from rats [169]. Still unresolved is the question of whether system y + consists solely of these proteins or involves other proteins as yet unidentified.…”