Neurochemical Research, Vol. 16, No. 3, 1991, pp. 215-226

The Structural and Functional Heterogeneity of Glutamic Acid Decarboxylase: A Review* M a r k G. E r l a n d e r 1 a n d A l l a n J. T o b i n 2,3,4,s

(Accepted January 1, 1991)

Studies of the GABA-synthetic enzyme glutamate decarboxylase (glutamic acid decarboxylase; GAD; E.C.4.1.1.15) began in 1951 with the work of Roberts and his colleagues. Since then, many investigators have demonstrated the structural and functional heterogeneityof brain GAD. At least part of this heterogeneity derives from the existence of two GAD genes. KEY WORDS: GAD; eDNA; GABA; pyridoxal phosphate; glutamate decarboxylase.

ANTIBODIES TO PURIFIED GAD ALLOWED THE ESTABLISHMENT OF GABA AS A NEUROTRANSMITTER IN MAMMALIAN BRAIN

Glutamate decarboxylase (glutamic acid decarboxylase; GAD; E.C.4.1.1.15) catalyzes the production of GABA, a molecule unmatched by any other known inhibitory neurotransmitter in its widespread distribution within vertebrate brain. Since Eugene Roberts was responsible for this laboratory's original interest in GABA, it is a particular pleasure to contribute to this Festschrift. Indeed, Roberts may claim direct and indirect paternity for much of the work that we review here. Roberts and his colleagues discovered GAD in the vertebrate brain some 40 years ago. In this review we trace the evolving conception of GAD's molecular identity from its original discovery in 1951 to our own recent work, which shows that two forms of GAD, with distinctive enzymatic properties and subcellular distributions, derive from two genes.

The discovery and subsequent establishment of GABA as a major inhibitory neurotransmitter in invertebrates and vertebrates followed a familiar pattern: the statement of an hypothesis, the application of a new technique, the investment of prodigious effort, and the shining of good fortune. Although GABA was first identified in the late nineteenth century, it was not until 1950 that Roberts and others demonstrated its importance in central nervous system function. GABA and GAD are Discovered in Vertebrate Brain. In 1950, Roberts (1) and Awapara (2), using a then new technique of two-dimensional paper chromatography, independently identified GABA as the previously unknown ninhydrin-reactive spot found exclusively in animal brains. Udenfriend then analyzed a sample provided by Roberts and Frankel (1) and independently confirmed its identity as GABA (3). Both Roberts and Awapara demonstrated the existence of an enzyme in mouse brain that was able to decarboxylate L-glutamate to form GABA. They suggested that its action might resemble that of the GAD already known to exist in bacteria and plants. During the next several years, Roberts and colleagues further characterized mouse GAD. They found

1 Neuroscience Program, 2 Department of Biology, 3 Molecular Biology Institute, and 4 Brain Research Institute, University of California, Los Angeles, Los Angeles, California 90024. 5 To whom correspondence should be addressed at the Department of Biology, University of California, Los Angeles, Los Angeles, CA 90024-1606. * In honor of the 70th birthday of Dr. Eugene Roberts.

215 0364-3190/91/0300-0215506.50/0 9 1991 Plenum PuNishing Corporation

216

Erlander and Tobin

(a) that active GAD requires pyridoxal 5'-phosphate (PLP), indicating that GAD can also exist in an inactive apoenzyme form, as well as an active holoenzyme (4); (b) that L-glutamate is indeed GA~'s primary substrate (5); (c) that the brain contains significantly more GAD activity than peripheral tissues (5); and (d) that rats receiving vitamin B 6 deficient diets have lower levels of holoGAD but not apoGAD (6). In 1953, Roberts (7) and others (8) established that GABA and c~-ketoglutarate undergo a reversible transamination to form glutamate and succinic semialdehyde in brain and liver. These findings laid the foundation for numerous future investigations which examined the role of the GABA shunt in intermediary metabolism, as proposed by Roberts in 1956 (9). (For review, see 10). Figure 1 shows the molecules and reactions of the GABA shunt. This early work also showed that GAD, unlike other enzymes involved in GABA metabolism, is primarily restricted to the nervous system of animals. Recent work, however, has demonstrated that low levels of GAD activity and of GAD mRNA are present in peripheral tissues, as well as in the brain (11). GABA Obtains Neurotransmitter Status. During the years 1956-57, interest in GABA increased significantly as a result of findings in several laboratories. Hayashi and Nagai (12) showed that GABA inhibits convulsions when applied to mammalian cortex; Killam (13,14) demonstrated that GAD activity and GABA levels were decreased in the brains of animals that had undergone semicarbazide-induced seizures; and Bazemore et al. (15) documented the identity of GABA as "Factor I," a molecule extracted from brain and spinal cord that inhibited the generation of impulses by the stretch receptor neuron of the crayfish. These observations were the impetus for a number of experiments which documented GABA as a neurotransmitter. The establishment of GABA as a neurotransmitter began with the demonstration by Kuffler and Edwards (16) that GABA duplicates the action of a naturally pro-

/ HOOCCH;CH;CH

NH; COOH

Glutamate

HOOCCH:CH~CH:N H: GABA

duced inhibitory transmitter by blocking sensory discharge of the crustacean stretch receptor. Kravitz and his Harvard colleagues (who included Zach Hall, Stephen Kuffler, Perry Molinoff, and David Potter) then completed a series of experiments which established GABA as an inhibitory neurotransmitter at the crustacean neuromuscular junction. The Harvard group showed (a) that GABA is the most potent of 10 inhibitory amino acids isolated from lobster in inhibiting excitatory junctional potentials on the opener muscle of crayfish leg (17); (b) that the molar ratio of GABA in inhibitory versus excitatory neurons of the lobster is 100:1 (18,19); (c) that GAD activity is ten times higher in the inhibitory versus excitatory neurons, while other enzymes of the GABA shunt (i.e., GABA transaminase and succinic semialdehyde dehydrogenase) are not preferentially concentrated (20); (d) that GABA is removed from the postsynaptic cleft by an uptake process (21); and (e) that the inhibitory nerve of the opener muscle of the claw released GABA when stimulated (22). In addition, Takeuchi and Takeuchi (23) showed that the convulsant picrotoxin antagonizes the action of GABA and the natural inhibitory transmitter similarly in the crustacean neuromuscular preparation. Molinoff and Kravitz (24) then partially purified GAD and showed that unlike mammalian GAD, GABA inhibited lobster GAD at a I~. of 1 mM. (For a review of the role of GABA in crustaceans, see 25). Subsequent work established GABA as an inhibitory neurotransmitter in the vertebrate CNS, as reviewed by Roberts (26). This demonstration was more difficult than in invertebrates because researchers could not identify GABA neurons among the plethora of morphologically distinct neurons in the CNS. In a recent review, Roberts (27) reflected on the frustration he felt at the time: "Interpretation of results from the above types of analyses [biochemical, electrophysiological, pharmacological, and iontophoretic studies] always suffered from the lack of definition, attributable to the presence of millions of cells of different types in any dissected re-

/tO HOOCCH:CH;C ~'COOH o- Ketoglutaratr

HOOCCH.,CH:CHO Succinic semialdehyde

SSA DH

,, HOOCCH2CH2COOH Succinic acid

Fig. 1. Reactions of the GABA shunt. Abbreviations for enzymes are as follows: Glutamate Decarboxylase is GAD; GABA Transaminase is GABA-T; Succinic Semi-Aldehyde Dehydrogenase is SSADH. Reprinted with permission from Raven Press.

Glutamic Acid Decarboxylase: A Review gion, and definitive conclusions were not possible about specific synaptic connections." Therefore, in 1968, Roberts decided, as he later said, to "go for broke" and embark on the difficult journey of purifying vertebrate GAD to homogeneity (27). GAD is Localized in Nerve Terminals. In 1973, Roberts, together with Jen-Yeng Wu and T. Matsuda, used 9,000 mouse brains to purify GAD 700-fold by conventional protein separation techniques (28). They reported the molecular weight of GAD to be 85,000 --2,000 by high speed sedimentation equilibrium analysis. Treatment of GAD with 6 M guanidine-HCl caused GAD to dissociate into two physically indistinguishable subunits of Mrs of 44,000 _ 2,000. In addition, the major component of purified GAD (there were also minor and faint bands) had a molecular size of 60,000, as estimated by its mobility in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (29). To reconcile the major, minor, and faint bands with sedimentation equilibrium and denaturation studies, Roberts and his colleagues suggested that native GAD may be composed of six identical subunits, each with a molecular size of 15,000 (29). The Roberts group then produced a rabbit antiserum to GAD (30). In 1974, in collaboration with the group of James Vaughn (also at the City of Hope) they reported the first visualization of GAD immunoreactivity in mammalian brain. This report was especially important for several reasons: (a) the demonstration of GAD immunoreactivity in the terminals of Purkinje neurons was final proof that GABA acted as an inhibitory neurotransmitter in those neurons; (b) their antisera allowed Roberts and Vaughn (32) to demonstrate unequivocally by electron microscopy the presence of GAD in close association with synaptic vesicles; and (c) the GAD antiserum would allow the mapping of GABA neurons throughout the vertebrate CNS (33-35).

GAD IS NOT A SINGLE MOLECULAR ENTITY. In 1970, Roberts and coworkers reported evidence for a second GAD, which they called GAD II. GAD II, they reported, was a non-neural, rnitochondrial form found in the brains, kidneys, and glial cells of chick embryos (36,37). Several years later, the existence of GAD II came into question when it was discovered that the 14CO2 evolved in assays of GAD II activity resulted from nonspecific CO2 release from contaminants of the purchased 14C-glutamate. (For additional details, see 38,39,40). Although this second GAD was, in this instance, artifactual, the idea has persisted that GAD may be not a single molecular entity. The question of molecular bet-

217 erogeneity has again arisen in recent biochemical and molecular studies of GAD, as we will discuss below. GAD is a Dimer. Since the initial purification of mouse GAD by Roberts and coworkers, several groups have reported the purification of GAD from the brains of rats (41-44), pigs (45,46), cows (47), and humans (48). Almost all of these reports lead to the following general consensus regarding GAD's molecular identity in all the mammalian species examined: (a) native GAD is a dimer of approximate Mr of 120,000; and (b) GAD is composed of two subunits which have molecular sizes in the range of 59,000-70,000. This consensus of GAD subunit size also matches the major polypeptide isolated from the initial GAD purification from mouse brain. Therefore, the minor and faint bands that also were present in the SDS-PAGE of purified mouse brain GAD may not be related to GAD subunit structure. Among the groups actively studying the biochemistry and enzymology of GAD, only Wu and his colleagues appear to hold views that diverge from this consensus. Wu and his coworkers agree that native GAD has a molecular size of about 120,000, but in 1986 they proposed that the active enzyme consists of two subunits with Mrs of 40,000 and 80,000 (43). In 1987, however, they reported a GAD in rat brain with two apparently identical subunits with Mr = 67,000 and common epitopes with their other reported GAD from (49). This latter GAD, then, has properties consistent with those isolated in other laboratories. Not all GAD Polypeptides are Identical. In addition to purifying GAD, many investigators have also generated antisera and monoclonal antibodies to GAD. Most of these immunological reagents have been used to detect the two subunits of GAD separated on immunoblots ("western blots"). We have summarized the published findings from such analyses in Table I. These data confirm the conclusions of biochemical studies of purified GAD: in all mammalian species examined, GAD is composed of two polypeptides of Mrs 59,000-67,000. Current estimates of the sizes of GAD polypeptides are derived from SDS-PAGE, which have given slightly different results in different laboratories. Thus the smaller GAD polypeptide is reported to have an Mr of 59,000 (44,50) and 60,000 (51), while the larger has reported Mrs of 62,000 (50), 63,000 (44), or 66,000 (51). Furthermore, the data from the western blots demonstrates that the two subunits of GAD have both common and unique epitopes (30,44,52-55). In addition, Legay et al. (56) have demonstrated that a significant fraction of native GAD is a homodimer. Thus, the two GAD polypeptides, with differing size and antigenicity, are likely to form two distinct homodimers. We find it difficult to

218

Erlander and Tobin Table I. GAD Antisera/Antibody I

I

Description of An!ibody/Antisera(Ab/As) Original Name of Type of Purified/Raised Paper Ab/As Ab From/In Matsuda/ Roberts (30)

Oertel/ Kopin (52,53)

Polyclonal

$3

Spink/ Martin (46) Chang/ Gottlieb (44)

GAD-6

Polyclonal

Mouse/Rabbit

Rat/Sheep

Polyclonal

Pig/Sheep

Monoelonal

Rat/Hybridoma

Source of Extract*

Data from Western Blots Number of Size of Polypeptides Polypeptides

Cat

Ref.

60, 66

(51)

Recombinant fusion-GAD(cat)

66

(51)

Quail, Rat, Mouse, Human, Rabbit, Monkey

59/62

(50)

Mouse, Cat & Human

60, 66

(51)

Recombinant fusion-GAD (Cat)

66~

(51)

Recombinant non-fusion GADs (rat)

65", 67"

(65)

62

(55)

60 66

(42, 46) (51)

Rat

59 only

(44)

Rat

65~ only

(65)

Mouse

658 only

(55)

40,80

(57)

1

80

(57)

Recombinant fusion-GAD (mouse) Pig & Rat Recombinant fusion-GAD(cat)

Wu/Lin (57)

Polyclonal

Rat/Rabbit

Wu/Lin (57)

Monoclonal

Rat/Hybridoma

6799

Polyclonal

Recombinant mouse GAD/ rabbit

Mouse

2

59, 62

(54)

K2

Polyclonal

Recombinant feline GAD/ rabbit

Mouse

1

67 ~

(55)

Rat

1

67

(65)

Katarova/ Greenspan (54) Kaufman/ Tobin (55)

Partially purified rat GAD

*Unless otherwise stated, source is brain homogenateof specified animal species. Recombinantmeans that GAD cDNA was expressed to generate GAD protein in bacterial or eucaryotic cell lines.

compare the results of Wu and colleagues (57) with others summarized in Table I because they do not show how their sera and monoclonal antibodies react on immunoblots with whole brain homogenates.

Two Genes Encode Distinct GAD Polypeptides. A major question remained unanswered: What is the molecular identity of the two GAD polypeptides? The application of molecular cloning techniques has been instrumental in answering this question. In 1986, this laboratory reported the immunological

"Size based on amino acid sequence ~om cDNA nucleotide sequences.

identification of a cDNA, within a kgt-11 bacterial expression library, that encodes cat brain GAD (51). We demonstrated the identity of the clone by a direct functional assay: the fusion protein made by k-GAD in E. coli lysogens had GAD enzymatic activity, stoichiometrically converting glutamate to GABA and C Q . The fusion protein was also recognized by all the then available GAD antisera. We also documented by in situ hybridization (58) that the regional distribution in mouse brain of GAD RNA matches previous immunocytochem-

Glutamie Acid Deearboxylase: A Review

ical studies with GAD antisera. In 1987, Mallet's group followed by cloning GAD from rat brain using a similar approach (59). Using our feline GAD eDNA as a probe, two other groups have isolated GAD cDNAs from mouse (54) and Drosophila (60). Amino acid sequences deduced from the nucleotide sequences of these cDNAs show that vertebrate GADs share more than 90% sequence identity. These three GAD genes are therefore likely to be homologous, that is, to derive from a common ancestral GAD gene. Even Drosophila GAD is recognizably similar to the mammalian GADs (60). An important question remained, however: which GAD subunit had we and others cloned? The answer to this question became evident with the generation of antibodies that were specific to one or the other GAD polypeptide. In 1988, Chang and Gottlieb (44) reported the generation of a monoclonal antibody (called GAD-6) which was only immunoreactive in western bIots with the smaller polypeptide. This laboratory also produced a polyclonal antiserum (called K-2) by injecting rabbits with the feline GAD which was synthesized in a bacterial expression system (55). The K-2 antiserum, recognized the feline fusion GAD and the larger GAD polypeptide in western blots containing whole brain homogenates (55), but GAD-6 did not react with our fusion feline GAD protein (55). Katarova et aI. (54) found the same recognition pattern with the GAD produced from their mouse eDNA in bacterial or eukarotic expression systems. Although Mallet's group never expressed their rat GAD eDNA as a non-fusion protein, the close identity of amino acid sequence (95%) and predicted molecular size of their rat GAD clone (61) is highly similar to our published feline cDNA (62), and it is likely that the rat eDNA is a homolog of the feline GAD eDNA. All these cDNAs thus appeared to encode the larger GAD polypeptide, whose sequence predicts a Mr of about 67,000. A crucial part of the original question still remained, however: what is the molecular identity of the smaller GAD polypeptide? Several lines of evidence suggested that the smaller and larger GAD forms were encoded by the same gene in vertebrates. 1. eDNA cloning using the antiserum of Oertel et al (52,53) to screen bacterial expression libraries, yielded only one GAD-encoding sequence in both cats and rats (51,59). 2. GAD67cDNA hybridized to the same DNA restriction fragments in Southern blots under conditions of both high and low stringency (allowing for 30% mismatch with the cloned feline, human, and rat GAD67 cDNAs). 3. In 1988, Chang and Gottlieb purified the smaller

219

GAD polypeptide by immunoaffinity chromatography and preparative SDS-PAGE (44). They determined the amino sequences of several CNBr cleaved peptides totaling 97 amino acids (44). Alignment of this amino acid sequence with that deduced from the nueleotide sequence of our feline cCNA revealed several stretches of exact match (8, 10, and 12 amino acids in length) between the two sequences and an overall identity of 70% (44). Chang and Gottlieb (44) suggested three alternative explanations for reconciling these sequence discrepancies: (a) species differences (the rat cDNA sequence from Mallet's eDNA was not then available),~ (b) alternative splicing of the same gene, and (c) the existence of two different genes encoding the two polypeptides. Another possibility not mentioned was sequencing errors in either the amino acid or nucleotide sequence. Because the correct identification of an amino acid in peptide sequencing can sometimes be ambiguous, it is possible, as stated by Mallet and coworkers in 1990 (61), that these sequence differences were artifactual. The long stretches of amino acid sequence identity (for example, an 8 and 10 amino acid identity in a 20 amino acid stretch) of Chang and Gottlieb's peptides and the deduced feline GAD suggested that low stringency Southern blots should reveal a second gene if it existed (assuming codon bias among vertebrate species). Since 1986, when we began to distribute the eDNA to scores of laboratories, however, no one reported the finding of more than one eDNA or gene. 4. Greenspan and colleagues (54) demonstrated the presence of two GAD genes in mouse, but one turned out to be a pseudogene (63). This led Greenspan's group to favor the hypothesis that mouse contained a single gene with the two forms derived from alternative splicing of a single gene (54). FinalIy, Martin and coworkers reported that partial proteolytic cleavage of purified smaller and larger GAD forms with staphylococcus aureus V8 protease yielded peptide fragments of virtually identical size (64). They concluded that these two GAD forms were either identical or very closely related (64). Therefore, by 1989, evidence had accumulated that allowed the conclusion that probably these two GAD forms were from the same gene. Unfortunately, neither we nor others could prove this conclusion with positive experimental evidence, thus, forcing us to rethink our original hypothesis. Our subsequent work demonstrated that the two polypeptides derive from two mRNAs, which in turn are the product of two separate genes. We demonstrated these conclusions by the following set of experiments (65): we used degenerate oligonucleotides as primers for a polymerase chain reaction (PCR) with eDNA derived from

220

Erlander and Tobin

rat brain mRNA. These degenerate oligonucleotides encode two amino acid sequences that are identical in rat and cat GAD. From this we obtained two different populations of PCR products. These two PCR products allowed us to obtain longer eDNA clones from a rat hippocampal eDNA library. One of these cDNAs was a rat cognate of our original feline eDNA, which has an open reading frame of 593 amino acids, a calculated molecular size of 66,600 (GAD67)and a 95% identity to the amino acid sequence of feline GAD (65). The second eDNA (which matches the partial sequences of Chang and Gottlieb) has an open reading frame of 585 amino acids, a calculated molecular weight of 65,200 (GAD65), and 65% amino acid sequence identity with feline GAD. The GAD65amino acid sequence matches the partial sequence of rat GAD CNBr peptides in 96/97 amino acid residues (65). The nucleotide sequence identity between the two rat GADs is 65% within the coding region. A scatterplot of the two nucleotide sequences (Figure 2) reveals sequence conservation within carboxy-terminal half of the coding region, but strong divergence in the nucleotide sequences encoding the 100 amino-terminal amino acid residues--a pattern also seen at the amino acid sequence level (65). We also have unequivocal evidence that these two GADs are encoded by distinct genes: (a) a 17-nucleotide segment represents the largest contiguous sequence with exact identity between the two GAD cDNAs (65), (b)

the two cDNAs do not cross hybridize with each other or with each other's mRNAs at high stringency (0.2 X SSC, 0.1% SDS, 55~ (65), and (c) GAD65and GAD67 cDNAs do not cross hybridize with our isolated genomic clones encoding GAD67and GAD65respectively (unpublished results). But does the newly cloned GAD cDNA encode the lower molecular weight GAD subunit as seen in western blotting experiments? We have expressed rat GAD65 in bacteria and found the following: GAD65 runs at the same electrophoretic mobility in SDS-PAGE as the smaller GAD polypeptide in brain extracts. It also reacts similarly with immunological reagents, including Chang and Gottlieb's GAD-6 (65). We therefore propose that the two polypeptides seen on western blots be designated as GAD65 and GAD67.

The Enzymatic Activity of GAD65 is More Responsive to PLP than that of GAD67. The investigation of the interaction between GAD and PLP began when Roberts and colleagues demonstrated that mammalian GAD required PIP for enzymatic activity (4). Since then, many workers have reported a strong correlation between the occurrence of seizures and the depletion of synaptic GABA and GAD activity with PLP-antagonists and carbonyltrapping agents. (For reviews, see 66, 67, and 68). The exact relationship between PLP and GAD has undergone close scrutiny over the years9 Because pyridoxine administration reversed the seizures induced by some carbonyl trapping agents and by

./ 9

/ L / '/

GAD 65

, 9 J

8

/

9 "

/r J

"" ..

9

~

GAD 67

Fig. 2. Scatterplotcomparingnucleotidesequencesof GAD6s.

7

Glutamic Acid Decarboxylase: A Review

221

all PLP-antagonists (summarized in 26 and 69), most workers assumed that GAD had a much lower affinity for PLP than other PLP-requiring enzymes (66). This hypothesis was extended further by the finding that GAD activity in brain homogenates could be enhanced from two to five-fold (depending on brain region assayed) by the addition of 0.5-1.5 mM PLP to the incubation mixture (71-75). This view, however, changed radically when Miller et al. (76) demonstrated that GAD was still more than 83% saturated with PLP even following exhaustive dialysis. Martin and coworkers (77) have presented a mechanism, based on their kinetic studies with purified GAD, which, to a large extent, explains this apparent paradox. They showed that GAD-PLP-glutamate complex not only produces holo-GAD (active GAD, PLP bound) and GABA, but also forms apo-GAD (inactive GAD, no PLP bound), succinic semialdehyde, and pyridoxamineY-phosphate (PMP) through a transamination reaction. (See Figure 3.) Unlike PLP, PMP readily dissociates from GAD (77), and thus a continuous supply of PLP is required to restore the level of holoGAD. In whole brain homogenates, only about 50% of total GAD is saturated with PLP (55). Under depolarizing conditions, PLP associates with apo-GAD to form active holo-GAD and thus increases GABA production in nerve terminals with increased neuronal activity (78,79).

PIC - 0 ENZ r N

HIC+NHa

\

S' " I ~ c ~ o ' ~ y

I NH~ Pyridoxlmi~

+ " +CO~-" CH~

I

Al:~lnzymG

11'

It

Gh/llmlll~

COl-

ENZ

H~ - N ~

I I s Z

CO~ ~H2 ~H~

HC'N-dH

!H, N~ 4-Ami~ ~Jtvrltl

Su~m~c llmillde~ydl

CO2ENZ CH= ~lH1 CH2

Altvn,ltiv,i

ENZ

CO+CH1

HC.N. CH

[NZ 9

3

CO2CH,

-,,~N-CH z

J,~,J

-r-Ammc~l~lv~te

Fig. 3. Catalytic cycle of brain GAD including the alternative transamination pathway as proposed by Martin and colleagues (75). Reprinted permission from Biochemistry Journal.

The amount of GABA available for synaptic release therefore depends on the interaction of GAD and PLP. This relationship suggests that PLP is an important regulator of GAD activity and thus of GABA synthesis. But this leads to two questions: do GAD65 and GAD67 interact differently with PLP, and if so, which form is responsible for the observed apo-GAD? Evidence for two GAD forms, distinguished by their differing interactions with PLP, was first described by Bay6n, Tapia and coworkers in 1977 (80-82). Their enzyme kinetic studies suggested that two non-interconvertible GAD forms exist in rat brain, one dependent and the other independent of free PLP. In addition, Dennor and Wu (73) have shown that the PLP-dependent and PLP-independent GAD forms have different molecular sizes and regional distributions in rat brain. On the basis of these reports, the laboratory hypothesized that GAD6s and GADa7 were, respectively, the PLP-dependent and PLP-independent forms. To test this hypothesis, we measured GAD activity in the supematants of brain homogenates after selective removal of GAD65 or GAD67 by immunoprecipitation (55). We found that GAD65 makes up most of the apo-GAD reservoir in GABA neurons (55). To further substantiate this finding, we subcloned both GAD65 and GAD67 cDNAs into vectors that allow their expression in bacteria (65). We were able to show the following: (a) both GADs catalyzed the conversion of [1-x4C]-glutamate to GABA and t4CO2 at specific activities comparable to or greater than brain homogenate, and (b) the addition of PLP stimulated GAD+s activity nearly seven-fold while GAD67 activity was only increased two-fold (65). This greater stimulation probably reflects the faster cycling of GAD65 through the inactivation cycle proposed by Martin and coworkers (77). This faster cycling suggests that GAD65contributes more to the pool of apo-GAD that exists in-vivo (83). Martin and coworkers (84) have confirmed this conclusion through a different experimental approach. After incubation of brain homogenates with 32P-PLP and then reduction with sodium borohydride, they have demonstrated that a significantly greater (about 10-fold) labeling by 32P-PLP of GAD65 than GAD67occurs. How this cycling mechanism would work in vivo is still unknown. Martin's group has reported that at low PLP concentrations (Ko for GAD:PLP is 10 -7 M), the transamination reaction is virtually undetectable (85). How, then, does such a large build-up of apoGAD occur? The simplest explanation wouId be that PLP and apoGAD are separated from each other, perhaps in different subcellular compartments or by differential interactions with other molecules. One possible explanation

222

derives from the observation that poIyanions stabilize apoGAD (86). ApoGAD, while formed in small amounts may be stabilized or tethered to polyanions and thus inhibited from interacting with PLP. Of this stabilized apo-GAD, a greater proportion would be GAD65because of its ability to cycle faster. When the nerve terminal requires greater amounts of GABA, this apoGAD is released (by an unknown mechanism via electrostatic interaction) and PLP becomes accessible to this apoGAD and forms holoGAD and thus increases GABA concentrations in the nerve terminal. GAD6s and GAD67Differ in Subcellular Location. In the course of characterizing GAD, investigators using subcellular fractionation have shown different subcellular distributions of enzymatically defined forms (39) and molecular sizes (87) of GAD. By using GAD-6 and K2, we have shown through immunohistochemical and subcellular fractionation experiments that more GAD65 is in nerve endings than in cell bodies and dendrites, while the opposite is observed for GAD67(55,65). In vivo experiments utilizing inhibitors of GABA synthesis and degradation have suggested that the GABA pool in neuronal cell bodies is different from that in the nerve terminals (88). Thus it is possible that GAD65 is mostly responsible for maintaining the GABA pool in nerve endings while GAD67 contributes more to the formation of GABA in dendrites and cell bodies and thus is more involved in cellular metabolism (through the GABA shunt). This hypothesis predicts that neurons with extensive dendrite arborizations and no axons would contain a greater amount of GAD67than GAD65. Supporting this hypothesis, we have found that the granule cells of the olfactory bulb, which form dendrodendritic synapses with mitral ceils (89), have 3-4 times more GAD67RNA than GAD65RNA (90). This distribution is consistent with the reported finding that most GAD activity in the olfactory bulb is present in $2 (supernatant of all cell bodies) and P1 (crude nuclear pellet) and not in synaptosomes (91). The role of these two GAD forms may not be so simple. For example, while GAD67is more concentrated in cell bodies and dendrites, GAD67is also present in nerve terminals (55). We therefore suggest that differential gene expression and subceUular location within a given GABA neuron are the two distinct contributions that can alter the amounts of GAD65and GAD67 present in the synapse. Neurons that phasically and rapidly produce large amounts of GABA may therefore contain a greater pool of PLP-inducible apo-GAD65. GABA produced by GAD67 in a nerve terminal, however, may reduce this inducibility, since nearly all the GAD67will be active holoenzyme.

Erlander and Tobin

FUTURE DIRECTIONS Although the molecular identity of GAD in vertebrate brain is now known, many unanswered questions about GAD remain. For example, establishing the molecular identity of GADs found in peripheral tissues, such as pancreatic B-cells (92), kidney (93), and oviduct (94; for review see 11) needs to be completed. Another issue is to test the hypothesis, set forth over 20 years ago, that the GAD gene is the primary lesion in the seizure disorder pyridoxine dependency (95; for review see 96). Undoubtedly, however, the most important issue is the regulation of GABA synthesis in vivo. We will first discuss what is already known and then suggest future investigations that could yield further information. How is GAD Activity Regulated? In addition to PIP, other molecules can modulate GAD activity (85). Martin and coworkers have demonstrated that ATP (or any polyanion) (86), glutamate (97), aspartate (98), and GABA (99) promote apoGAD formation, while inorganic phosphate (100) and PLP (100) promote holoGAD formation (for review see 85). Furthermore, these effectors act differentially on the three GAD forms (called peaks I, II, and III) that Martin and coworkers have isolated by adsorption chromatography on phenyl-Sepharose (42). The relationship between the GAD forms described by Martin and his colleagues and the two forms we have cloned (GAD65 and GAD67) is beginning to be understood. Martin and coworkers have designated the molecular weight of GAD65 in their western blots as a 63 kD polypeptide (102). Peak III contains predominantly the 63 kD GAD polypeptide (42). Thus, peak III is enriched with GAD65. The molecular identity of 54 kD and 59 kD GAD polypeptides found in peaks I and II remains to be determined. Neither our own experiments nor work in other laboratories have revealed significant amounts of GAD polypeptides with molecular weights in the 54 kD to 59 kD range in whole brain homogenates (44,50,55,65). In contrast, Chang and Gottlieb (44) demonstrated that affinity-purified rat GAD contains several polypeptides at 55 kD in addition to a 59 kD and 63 kD polypeptide (all of which were immunoreactive with the antiserum of Oertel, Kopin, and their colleagues and that of Matsuda, Roberts, and their colleagues). Whether the 54, 55, or 59 kD GAD polypeptides observed are degradative products or represent functional molecules in vivo is still uncertain. Uncertainty of molecular identity is a major obstacle in interpreting the enzymological studies of GAD. Expressing the cDNAs of GAD65 and GAD67 in bacterial or eukaryotic cell lines should eliminate many of the

Glutamic Acid Decarboxylase: A Review

previous problems that have plagued these studies and thus allow a better dissection of GAD enzymology. How Do Cells Accomplish the Differential Subcellular Distribution of GAD65 and GAD677A clue to answering this question may lie in understanding how GAD is transported to the nerve terminals. In an elegant study using electron microscopy, Roberts and coworkers (103) demonstrated in 1975 that GAD is in close association with small vesicles in the axons of growing neurites of the developing rat cerebellum. Thus, the differential distribution may be governed by a differing interaction of GAD65 and GAD67with synaptic vesicles. In 1968, Fonnum reported that GAD could become associated with membranes in the presence of CA + + (104). In addition, Tapia and coworkers (105, 106) demonstrated that this Ca + § induced binding only occurred with negatively charged liposomes (i.e., phosphatidyl serine) and that the PLP-dependent GAD was the predominantly bound GAD form. Because high concentrations of CA + + (0.25 mM to 5 raM) are needed for this binding to occur, it seems unlikely that this electrostatic interaction by itself represents a physiological mechanism (104-106). More recent findings, however, have shed some light on the issue of electrostatic interaction. Martin and coworkers (86), for example, have demonstrated that apoOAD has a strong affinity for polyanions. For example, the KD for the polyanion inositoI hexasulfate is approximately 50 nM (86). This may have physiological significance because the cytoplasmic face of synaptic vesicles is known to be enriched in acidic phospholipids (107). In addition, Chang and Gottlieb (44) showed that 60% of GAD is membrane bound and can be solubilized with 0.2% Triton-X-100 but not with high salt (1 M NaCI or 1 M KCI). They suggest that a significant amount of GAD interacts with membranes through a hydrophobic interaction. Thus, previous investigations have documented that GAD has the capability to interact with membranes by hydrophobic or electrostatic mechanisms. Proteins can also associate with membranes through fatty acylation. Specifically, fatty acylation of proteins can occur by covalent modification via esterification, Nmyristylation, and glypiation (C-terminal glycosyl phosphatidylinositol attachment). (For review, see 108). The unifying feature of all known N-myristylated proteins in the presence of a N-terminal glycine (108). Both GADs contain a N-terminal alanine (65); thus N-terminal myristylation probably does not occur to either GAD. Proteins undergoing glypiation contain N-terminal signal sequences and a stretch of 20-30 amino acids in the Cterminal of primarily hydrophobic residues (I10). Neither GAD fits the general consensus of glypiated proteins (65). Furthermore, glypiated proteins are not easily sol-

223

ubilized by Triton-X-100 (111), while membrane GAD is (44). Covalent modification of cysteine residues by esterification most likely occurs when three hydrophobic residues precede the cysteine residue (t12). Neither GAD contains this consensus sequence (65). There do not appear to be any known consensus sequences for serine or threonine based esterifications, thus, whether this covalent modification occurs in GAD65 or GADa7 will have to be determined empirically. The differential distribution of GAD6s and GAD67 in GABA neurons thus may result from differing interactions With vesicles. Other proteins do interact specifically with synaptic vesicles. For example, synapsin I, a small vesicle-associated protein, interacts with phospholipid bilayers through electrostatic and hydrophobic interactions (113). GAD65 may have a greater propensity of such electrostatic interactions because more GAD65 than GAD67exists in apoGAD form in vivo. Thus, the kinetic characteristics of the two GADs may dictate differential localization. How is GAD RIgA Regulated In-l/Tvo? Lesioning of the striatal-nigral pathway (114) or the inferior oliveclimbing fiber projection (115) causes an increase of GAD activity and of GAD67 RNA in the striatum and Purkinje cells respectively. Lesioning of the climbing fibers is known to increase the firing rate of Purkinje celIs (116,i17); thus an increase in neuronal activity has been strongly correlated with increased GADs7 RNA concentrations. We have shown that GAD67 RNA concentrations are increased in GABA neurons of rats stereotaxically injected with kainate in the CA3 hippocampal pyramidal field (118). The mechanism underlying these reported GAD67 RNA increases is unknown, but at least two possibilities exist: (a) increased stability of GAD6v RNA or (b) increased transcriptional rate of the GAD67gene. Nuclear run-on experiments should distinguish between these two possibilities. In either case, transient transfection experiments should delineate the RNA or DNA sequences responsible for increased RNA stability or transcriptional rate respectively (119). Splicing events during development also affect GAD production. Gottlieb and coworkers (120) have recently shown that the translation of active GAD67in rat embryo is regulated by a splicing event which inserts a 86 base pair exon containing a stop codon. This splicing event appears to be restricted to embryos and is not present in adult rat brain. Because Gottlieb's group has shown that this splicing occurs in two CNS derived cell lines (C6glioma and B65), experiments designed to identify what molecules underlie this mechanism can be more readily conducted.

224 Understanding the regulation of GABA production invivo requires consideration of both GADs. For example, rat cerebellar cortex contains both GAD65 and GAD67 mRNAs in the same neurons (Purldnje, Golgi II, stellate, and basket), but at dramatically different levels (90). Identifying the mechanisms responsible for both the coordinate and the separate regulation of the two GADs is important to understanding both how a neuron becomes GABAergic and how it regulates GABA production. In a 1986 review (26) Roberts compared his experience with GABA-related discoveries to peeling an onion, with "one problem inevitably leading to another in endless succession." Unlike an onion, however, knowledge of GABA regulation has a central core. And that core contains at least two GAD genes. REFERENCES 1. Roberts, E., and Frankel, S. 1950. Gamma-Aminobutyric Acid in Brain: Its Formation From Glutamie Acid. J. Biol. Chem. 187:55-63. 2. Awapara, J., Landua, A., Fuerst, R., and Seale, B. 1950. Free Gamma-AminobutyricAcid in Brain. J. Biol. Chem. 187:35-39. 3. Udenfriend, S. 1950. Identificationof Gamma-AminobutyricAcid in Brain by the Isotope Derivative Method. J. Biol. Chem. 187:6569. 4. Roberts, E., and Frankel, S. 1951. Glutamic Acid Decarboxylase in Brain. J. Biol. Chem. 188:789-795. 5. Roberts, E., and Frankel, S. 1951. Further Studies of Ghtamic Acid Decarboxylase in Brain. J. Biol. Chem. 190:505-512. 6. Roberts, E., Younger, F., and Frankel, S. 1951. Influence of Dietary Pyridoxine on Ghtamic Decarboxylase Activity of Brain. J. Biol. Chem. 191:277-285. 7. Roberts, E., and Bregoff, H. 1953. Transamination of GammaAminobutyric Acid and 13-Alaninein Brain and Liver. J. Biol. Chem. 201:393-398. 8. Bessman, S., Rossen, J., and Layne, E. 1953. Gamma-Aminobutyric Acid-Glutamic Acid Transamination in Brain. J. Biol. Chem. 201:383-391. 9. Roberts, E. 1956. Formation and Utilization of Gamma-Aminobutyric Acid in Brain. Pages 11-25, in Korey, S. (ed.), Progress in Neurobiology, Vol. 1, Hoeber-Harper, New York. I0. Baxter, C. 1976. Some Recent Advances in Studies of GABA Metabolism and Compartmentation. Pages 61--87, in Roberts, E., Chase, T., and Tower, D. (eds.), GABA in Nervous System Function, Raven Press, New York. 11. Erdo, S. and Wolff, J. 1990. Gamma-Aminobutyric Acid Outside the Mammalian Brain. J. Neurochem. 54:363-372. 12. Hayashi, T. and Nagai; K. 1956. Action of w-amino acids on the Motor Cortex of Higher Animals. Especially Gamma-AminoOxy-Butyric Acid as the Real Inhibitory Principle in Brain. Proe. XXth Int. Physiol. Congr., 410. 13. Killam, K. F., and Bain, J. A. 1957. Convulsant Hydrazides 1: In Vitro and In Vivo Inhibition of Vitamin B 6 Enzymes by Convulsant Hydrazides. J. Pharmacol. Exp. Ther. 119:255-262. 14. Killam, K. F. 1957. ConvulsantHydrazides2: Comparisonof Electrical Changes and Enzyme Inhibition Induced by the Administration of Thiosemicarbazide.J. Pharmacol. Exp. Ther. 119:263-271. 15. Bazemore, A. W., Elliott, K. A. C., and Florey, E. 1957. Isolation of Factor I. J. Neurochem. 1:334-339. 16. Kuffler, S., and Edwards, C. 1958. Mechanism of Gamma Aminobutyric Acid (GABA) Action and its Relation to Synaptic Inhibition. J. Neurophysiol. 21:589-610. 17. Dudel, J., Gryder, R., Kaji, A., Kuffler, S. W., and Potter, D.

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