Several laboratories have studied the cellular localization of NAAG in the brains of a number of species using immunocytochemical methods. The immunogen has routinely been NAAG linked by to a carrier protein by carbodiimide. To minimize cross-reacticity with NAA, polyclonal antibodies purified by affinity chromatography, 13'14 or a highly specific monoclonal antibody has been selected.15 Consistent with the uneven regional distribution of
NAAG levels, NAAG expression is restricted to discrete subsets of neurons. For example, intense immunoreactivity is noted in the retinal ganglion cells and optic nerve, motor neurons and their axon and terminals, locus ceruleus noradrenergic neurons, GABAergic interneurons in the hippocampus and the subhuman primate and human cortico-limbic pyramidal cells13'14,16-19 Ultra-structural studies demonstrate the localization of NAAG in storage vesicles,20 consistent with its calcium-dependent evoked release upon stimulation of NAAG containing projections such as the retinal-tectal pathway.21' 22
The diverse co-localization of NAAG with different neurotransmitters including glutamate, GABA, acetylcholine and norepinephrine16 is similar to that of other neuropeptides, which also co-localize with more than one neurotransmitter system. Nevertheless, this diverse co-localization, especially in motor neurons,23 raises important but as yet unanswered questions about the role of NAAG in modulating neurotransmission.
The physiologic effects NAAG have been somewhat controversial. The initial finding of excitatory effects in the olfactory bulb7 were likely the result of contaminating K+ and/or hydrolysis to free glutamate.24 Other studies suggested a weak, agonist effect of NAAG at NMDA receptors.25-27 Recent studies, however, indicate that NAAG antagonizes NMDA receptor responses in a glycine reversible fashion at the Shaffer collateral-CA1 pyramidal cell synapse.28 In addition, Wroblewska et al.29 screened the effects of NAAG in CHO cells expressing the different members of the mGLUR family and showed that NAAG is a full and selective agonist at mGluR3. This important discovery has been confirmed in the olfactory bulb with electrophysiological methods.30
Riveros and Orrego,31 using partially purified synaptic membranes, were the first to report the hydrolysis of NAAG to yield glutamate. Blakely et al.32 subsequently demonstrated that this hydrolysis was potently inhibited by quisqualic acid. Using this criterion, Slusher et al.33 purified to homogeneity a 94 kda plasma membrane glycoprotein that possessed this quisqualate-sensitive peptidase activity, which glycoprotein is now known as Glutamate Carboxy Peptidase II (GCPII; EC 18.104.22.168). With antibodies raised against the homogeneously pure protein, Carter et al.34 isolated a rat brain cDNA encoding GCPII. Sequence analysis indicates that it is a type II membrane protein in the M28 peptidase family with a short intracellular tail and a transmembrane domain for amino acids 20 through 43. Site-directed mutagenesis has clarified both the catalytic site of the enzyme and the location of the amino acids binding zinc.35 Molecular studies demonstrate that GCPII is identical to folate hydrolase in the gut36 and to prostate specific membrane antigen (PSMA),34, 37 a cell surface protein highly expressed on metastatic prostate cancer cells.
Immunocytochemical as well as in situ hybridization studies demonstrate that GCPII is selectively expressed in astrocytes in the brain with an uneven regional distribution.38, 39 Particularly high levels of expression are observed in the Bergmann glial cells. It is also expressed in non-myelating Schwann cells in the periphery, including those surrounding motor neuron terminals at the end plate.40 In the periphery, GCPII is expressed in the testicles and in the tubules of the kidney. However, the exclusive role of GCPII in the hydrolysis of NAAG has been called into question by studies in mice homozygous for a null mutation of the GCPII gene. The brains of these mice exhibit a residual of 15% GCPII-like enzyme activity with no alterations observed in the levels of NAAG, NAA, glutamate or aspartate.41 Recently, a second gene with high homology to GCPII has been cloned.42, 43
Many substantive unanswered questions remain with regard to the role of NAAG and its disposition in the nervous system. The mechanisms responsible for its synthesis remain obscure, although it is likely synthesized by an enzymatic and not an mRNA dependent process. Since GCPII is expressed in astrocytes, it is unclear what determines the levels of
NAAG in neurons. Given the absence of a behavioral phenotype with the GCPII knock-out mice, the role of NAAG as a co-transmitter in a variety of neuronal systems that do not appear to use glutamate as their primary neurotransmitter remains to be determined.
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