A Human Tyrosine Hydroxylase Isoform Associated with Progressive
Supranuclear Palsy Shows Altered Enzymatic Activity*
Sylvie
Bodeau-Péan
,
Philippe
Ravassard
§,
Martin
Neuner-Jehle
,
Baptiste
Faucheux¶,
Jacques
Mallet
, and
Sylvie
Dumas
From
Laboratoire de Génétique Moleculaire
de la Neurotransmission et des Processus Neurodigènératifs,
CNRS UMR 9923 and ¶ INSERM U289, Hôpital de la
Salpêtrière, 75013 Paris, France
 |
ABSTRACT |
A novel human tyrosine hydroxylase (HTH)
messenger RNA subgroup generated by alternative splicing and
characterized by the absence of the third exon was recently identified.
The corresponding putative protein lacks 74 amino acids including
Ser31 and Ser40, two major
phosphorylation sites implicated in the regulation of HTH activity.
These mRNA species are detected in adrenal medulla and are
overexpressed in patients suffering from progressive supranuclear palsy, a neurodegenerative disease mostly affecting catecholaminergic neurons of the basal ganglia.
In the present work, an HTH protein isoform lacking exon 3 was
identified in human adrenal medulla. For this purpose, an antibody was
raised against the HTH exon 3. The effect of the removal of exon 3 on
the enzymatic activity of HTH was studied in vitro by comparing a purified recombinant fusion protein without exon 3 (glutathione S-transferase (GST)-HTH
3) to the equivalent
protein containing exon 3 (GST-HTH3). In initial velocity conditions, GST-HTH
3 has 30% of the maximal velocity of GST-HTH3. Moreover, the
skipping of exon 3 results in the absence of activation of GST-HTH by
heparin and increases by 10-fold the retroinhibition constant for
dopamine, demonstrating the involvement of exon 3 in the regulation of
HTH enzymatic activity. The identification of a variably expressed HTH
isoform that lacks an exon implicated in activity regulation supports
the view that HTH alternative splicing contributes to the functional
diversity within the catecholaminergic system and may be implicated in
some neurological diseases.
 |
INTRODUCTION |
Tyrosine hydroxylase
(TH)1 has been the subject of
extensive investigation, largely because it catalyzes the rate-limiting step in the biosynthesis of catecholamines. The regulation of the TH
level and of its enzymatic activity is thus a major mechanism for
controlling the amount of these important amines in blood and synapses.
TH is regulated by almost all possible mechanisms of transcriptional
and post-transcriptional control including regulation of the
transcription rate, alternative splicing of the premessenger, variable
stability of the mRNA, translational control, and modulation of the
enzymatic activity (1). Recently, it has been shown that some human TH
(HTH) gene sequence variants are associated with abnormal TH enzymatic
activity and may be involved in some neurodegenerative diseases.
Indeed, point mutations in the coding sequence of HTH have been found
that decrease the activity of the enzyme in patients with
L-DOPA-responsive dystonia (2) and inherited juvenile
L-DOPA-responsive parkinsonism (3). There is also emerging
evidence that alternative splicing of TH premessenger RNA may have
physiological and even pathological consequences (4, 5). Initially,
four HTH mRNA variants generated by alternative splicing were
cloned and characterized by (i) the differential use of two splicing
donor sites within exon 1 and (ii) the differential inclusion of exon 2 (4, 6, 7). All four HTH species are present in human catecholaminergic
tissues (8, 9). Interestingly, the amount of the species containing exon 2 (HTH-3 and HTH-4) is enhanced in pheochromocytoma, a tumor of
adrenal medulla (4). The four mRNAs encode proteins that differ in
the N-terminal regulatory region and present different enzymatic
activities (1, 10). These data suggest that alternative splicing of TH
premessenger RNA is significant and prompt us to further study the
diversity of HTH mRNAs. Recently, we identified previously
undetected HTH mRNA species lacking exon 3 in the adrenal medulla
(5). These novel variants are produced by the junction of the splicing
donor sites of exons 1a-, 1b-, or 2- directly to the acceptor site of
exon 4. By RNase mapping experiments, we have shown that exon 3 was
skipped in 4 to 6% of the total HTH mRNA population in normal
adrenal medulla, a proportion similar to that of the previously
described HTH-3 and -4 mRNA species (11, 12). These novel mRNA
species were unusually abundant (5) in adrenal medulla of patients
suffering from progressive supranuclear palsy (PSP), which is a severe
neurodegenerative disease predominantly characterized by an alteration
of the catecholaminergic neurons within the basal ganglia (13). The
novel mRNA species encode putative HTH proteins lacking the 74 amino acids of the exon 3. This region includes Ser31 and
Ser40, two phosphorylation sites that participate in the
regulation of HTH enzymatic activity (1). Consequently, it is likely
that the alternative splicing of exon 3 has consequences for the
regulation of catecholamine biosynthesis.
In the present work, we analyzed the expression of this novel HTH
isoform lacking exon 3 (HTH
3) in human adrenals by Western blotting
experiments. For that purpose, we raised an antibody against the HTH
exon 3. We also tested the effect of exon 3 skipping on the in
vitro enzymatic activity of HTH by the use of purified recombinant
HTH proteins in fusion with glutathione-S-transferase (GST). It is
clearly established that TH activity is positively regulated by
phosphorylation and by binding to any of several polyanions such as
heparin, polyglutamate, and phosphatidylserine, which change the
conformational state of the enzyme (14). Interestingly, the heparin
binding site on rat TH (RTH) has been described as being precisely
localized within an exon corresponding to HTH exon 3 (15). We therefore
tested the effect of exon 3 skipping on HTH activation by heparin and
show that GST-HTH
3 enzymatic activity is insensitive to this
molecule. Feedback inhibition by catecholamines is one of the major
mechanisms for modulating HTH enzymatic activity, and the N-terminal
part of the enzyme plays an important role in retroinhibition by
dopamine (16). Thus we tested the effect of dopamine on GST-HTH
3
activity and found that HTH exon 3 is required for feedback inhibition.
 |
EXPERIMENTAL PROCEDURES |
Construction of the Expression Vectors--
The complete coding
sequence of HTH3 was excised from PET3a (Stratagene) (10) by
NdeI-BamHI digestion. The fragment was blunted by
Klenow polymerase and transferred into the SmaI restriction site of PGEX-2T (Amersham Pharmacia Biotech). This plasmid permits the
production of HTH3 fused to Schistosoma japonicum GST. A
sequence encoding the corresponding isoform of HTH but lacking exon 3 (HTH
3) was obtained by deletion of exon 3 from PGEX-2T-HTH3 as
described in Lanièce et al. (17). Briefly, two
amplification products of the sequences corresponding to HTH exons 1 + 2 and exons 4 + 5 were joined by polymerase chain reaction, and the
final amplification product was used to replace the HTH3 sequence
between the BamHI and XhoI sites in PGEX-2T-HTH3.
The primers used were (i°) ccataatggatccATGCCCACCCCCGACGCC (capital letters, HTH sequence beginning with initiator codon; bold,
BamHI site) and GCTTCAAACGT-CTTGGGGTGGG (junction
exon2-exon 4, antisense) and (ii°) CCACCCCAAG-ACGTTTGAAGC (junction
exon2 -exon 4, sense) and CGGGTGGTCCAAGTCCAG (within exon 5). The
PET3a-HTH
3 construct was obtained from PET3a-HTH3 by deletion of the
third exon using the same method. All amplified sequences were checked by sequencing.
Purification of GST-HTH Fusion Proteins--
PGEX-2T-HTH3 and
-HTH
3 constructions were introduced into electrocompetent AX90
Escherichia coli cells. Production of the fusion proteins
(GST-HTH3 and GST-HTH
3) was induced by
isopropyl-1-thio-
-D-galactopyranoside, and the proteins
were affinity-purified on glutathione-agarose beads according to the
manufacturer's instructions (Amersham Pharmacia Biotech) with minor
modifications. E. coli cells were harvested at 25 °C
instead of 37 °C to avoid formation of inclusion bodies. After
elution from the glutathione-agarose beads, fusion proteins were
extensively dialyzed against phosphate-buffered saline at 4 °C, then
to desalt, concentrate, and eliminate low molecular weight protein
contaminants, fusion proteins were centrifuged on Microcon-100
microconcentrators (Amicon, 100-kDa molecular mass cut-off columns).
Protein purity was confirmed by electrophoresis on a 9% polyacrylamide
denaturing gel followed by Coomassie Blue staining. The protein
quantities were first estimated with the Bradford method, then
precisely measured by comparing the intensity of the Coomassie
Blue-stained band to a standard curve obtained with bovine serum
albumin (Image 1.59 software).
Measuring of GST-HTH Activities--
GST-HTH3 and -HTH
3
enzymatic activities were tested by a radioenzymatic assay as described
in Alterio et al. (10) with minor modifications. Briefly,
the enzymatic activities were measured as the release of tritiated
water from L-[3H]tyrosine at 30 °C in 0.1 M Hepes, pH 7, 40 µM tyrosine, 0.5 mg/ml
catalase, 0.1 mM FeSO4, 0.32 µCi of
L-[3H]tyrosine (Amersham Pharmacia Biotech)
following addition of the cofactor BH4 (250 to 3000 µM)
diluted in 20 mM dithiothreitol. Fusion proteins were
diluted before activity assay in 0.1 M Hepes, pH 7, containing 1 µg/µl bovine serum albumin. Each activity was measured
in triplicate, and further experiments were performed with several
independant enzyme batches.
We first established the initial velocity conditions in which the
activity increases linearly with time and enzyme quantity. Using
different BH4 concentrations (from 250 to 3000 µM),
Lineweaver-Burk curves were then traced for both isoforms with the
Kaleidagraph software. The experimental results were fitted for the
inhibition by excess of BH4 substrate (10) with the following equation: V = Vm/(Km + [BH4] + [BH4]2/Ki), according to
Dixon and Webb (18), where Vm is the maximal
velocity of the enzyme, Km is the apparent Michaelis
constant for BH4, and Ki the inhibition constant for BH4.
To analyze the regulation of GST-HTH3 and -HTH
3 by polyanions,
heparin (Calciparine Sanofi, France) was added to the assay mix (5 to
15 IU) before the addition of BH4 (500 µM). Similarly, final product retroinhibition was tested by the addition of dopamine (3-hydroxytyramin; Sigma) (10 to 100 µM) to the reaction
before the addition of BH4 (500 µM). The inhibition
constant for dopamine (Ki) was determined
graphically for both isoforms by plotting 1/V as a function of dopamine
concentration for 3 different BH4 concentrations (250, 500, and 1000 µM). The point of intersection of the 3 lines gives
Ki directly (Dixon method, see Ref. 18).
Synthesis of anti HTH-exon 3 Antibody--
The nucleotide
sequence corresponding to the 59 first amino acids of the third exon of
HTH was amplified by polymerase chain reaction with oligonucleotides
ccataatggatccCGGTTCATTGGGCGCAGG and
ccacaatgaatCCTCGGGGAGAAGAGCAG, purified, digested, and
introduced between the BamHI and EcoRI
restriction sites of pGEX-2T (Amersham Pharmacia Biotech). The
amplified products were checked by sequencing. The GST-HTHexon3 fusion
protein was produced in AX90 cells and purified according to the
manufacturer's instructions (Amersham Pharmacia Biotech). The fusion
protein was dialyzed against 20 mM Tris, pH 7.5, 0.1 M NaCl, 0.2 mM EDTA, 1 mM
dithiothreitol and sent to Eurogentec (Ougrée, Belgium) for
antiserum production in rabbits. The anti-HTHexon 3 antiserum was
purified by affinity using the GST-HTHexon3 antigen immobilized on
Immobilon-P membranes (Millipore) using standard methods. The
immunoreactivity and specificity of this antibody was checked by
Western blotting experiments with total lysate from BL21 bacteria
(Stratagene) transformed with PET-3a-HTH1 and PET-3a-HTH
3.
Western Blotting Experiments--
Frozen post-mortem human
adrenal glands from controls and PSP patients were obtained from the
brain bank of Inserm U 289 (Hôpital de la
Pitié-Salpêtrière, Paris) and from Dr. P. F. Plouin (Hôpital Broussais, Paris). PSP was clinically
characterized, and the diagnosis was confirmed as described in Dumas
et al. (5). Tissues were lyzed in 10 mM Tris, pH
7.5, 5 mM EDTA supplemented with 1 mM phenylmethyl sulfonyl fluoride; Sigma) and centrifuged for 10 min at
4 °C. The total protein concentration was estimated by the Bradford
method. Western blotting experiments were performed by standard
procedures with an anti-RTH polyclonal antibody provided by Dr. J. F. Reinhard (19) and 125I-protein A. The membrane was
stripped in 0.1 M glycine, pH 2.8, 0.05% Tween 20 at
80 °C and reprobed with the anti-HTHexon3-purified antiserum. The
amount of the HTH
3 isoform in the adrenal samples was estimated with
the Image 1.59 software.
 |
RESULTS |
Identification of an HTH Isoform Lacking Exon 3 in Human
Adrenals--
To test human tissues for the presence of an HTH protein
isoform lacking exon 3, we raised and purified an antiserum against the
third exon of HTH. We ascertained the immunoreactivity and specificity
of affinity-purified anti-HTHexon3 antibodies by Western blotting
experiments with HTH
3 (containing exons 1a,2,4-14) and HTH1
(containing exons 1a,3,4-14) produced in E. coli by the
PET-3a expression system. As expected, this anti-HTHexon 3 antibody
only recognized the HTH1 isoform (Fig.
1B), whereas a polyclonal
anti-RTH antibody (19) recognized both HTH isoforms (Fig.
1A), attesting to the specificity of the anti-HTHexon3
antibody.

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Fig. 1.
Immunoreactivity and specificity of the
antibodies affinity-purified from the antiserum directed against
HTH-exon 3. Whole bacterial lysates from E. coli cells
transformed with various PET 3a-HTH expression vectors, PET3A-HTH1
(1)and PET3A-HTH 3 (2), were run on a 12% polyacrylamide
denaturing gel, transferred to a polyvinylidene difluoride membrane,
and immunodetected with polyclonal anti-wholeTH antibody (A)
(19) or anti-HTHexon 3 purified antiserum (B).
|
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Then, to determine the expression profile of the HTH
3 isoform in
human tissues, total protein extracts from control and PSP adrenal
medullas were probed with both antibodies by Western blotting experiments. Recombinant HTH proteins expressed from the PET-3A system
in E. coli were used as size controls. With the polyclonal anti-rat TH antibody (19), several bands were detected in these tissue
samples (Fig. 2A). The two
slower migrating bands correspond, respectively, to HTH 3 and 4 and HTH
1 and 2, as described previously (20). Two other isoforms of
approximately 55 and 50 kDa were found in all tissue samples; they were
also detected in a noncatecholaminergic rat cell line (ST14A) stably
transfected with HTH1-cDNA, showing that were not the result of
alternative splicing.

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Fig. 2.
Presence of an HTH isoform lacking exon 3 in
human adrenal medulla samples. Western blotting experiments were
performed with 10 to 100 µg of total protein from
PET3a-HTH1-transformed E. coli cells (HTH1),
PET3a-HTH 3-transformed E. coli cells (HTH 3),
HTH1-cDNA-transfected eucaryotic cells (TC), adrenal
medulla from a PSP patient (P1), and adrenal medulla from
two controls (C1 and C2). The blot was incubated
with polyclonal antibody (19) (A), then stripped and
incubated with anti-HTHexon 3 purified antiserum (B). The
sizes of the different HTH isoforms are indicated. The proportion of
HTH 3 is 2, 1.5, 12, respectively, in C1, C2, and P1.
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|
Interestingly, as seen on Fig. 2A, the polyclonal anti-rat
TH antibody detected a novel protein of the same size as recombinant HTH
3 in the PSP adrenal medulla sample. To confirm the identity of
this isoform, the membrane was stripped and reincubated with the
anti-HTHexon3 antibody (Fig. 2B). The novel protein was not recognized by this antibody, evidencing that it lacks exon 3 and is
certainly HTH
3. This isoform is present in a substantial amount in
PSP adrenal medulla and is present in much lower amounts in normal
adrenal medullas (Fig. 2).
GST-HTH
3 Presents 30% of GST-HTH3 Maximal Velocity in
Vitro--
To analyze the involvement of exon 3 in HTH enzymatic
activity, the cDNAs encoding HTH
3 and HTH3 (the equivalent
isoform containing exon 3) were inserted into PGEX-2T (Amersham
Pharmacia Biotech), allowing the synthesis of GST fusion proteins. Both GST-HTH
3 and GST-HTH3 fusion proteins were produced and purified from total bacterial lysate on an affinity column. The integrity and
purity of both fusion proteins were ascertained by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (Fig.
3). The activities of the recombinant
GST-HTH proteins were measured by a radioenzymatic activity assay. The
absence of activity presented by GST alone in this assay was
ascertained (data not shown). The initial velocity conditions in which
product formation increases linearly with time and enzyme concentration
were determined (Fig. 4), and all further
experiments were performed in the following conditions: 50 ng of fusion
protein, 5 min reaction time. The GST-HTH activities were measured with
various BH4 concentrations from 400 to 3000 µM, and the
Lineweaver-Burk plots were traced, showing typical excess substrate
inhibition (Fig. 5). The kinetic parameters were calculated according to the classical analysis of
substrate inhibition kinetics, as described under "Experimental Procedures." The maximal velocities of GST-HTH3 and -HTH
3 were, respectively, 1400 (±70) and 450 (± 40) pmols of DOPA/min/µg of enzyme, which shows that GST-HTH
3 has approximately 30% GST-HTH3 maximal velocity. The absence of exon 3 did not significantly change
the Michaëlis constant (Km) of the enzyme for the cofactor BH4 (174 ± 24 µM for GST-HTH3,
180 ± 50 µM for GST-HTH
3) nor the inhibition
constant (Ki) for BH4 (4100 ± 500 µM for GST-HTH3, 2800 ± 500 µM for
GST-HTH
3).

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Fig. 3.
Purity and integrity of GST-HTH fusion
proteins. Coomassie Blue staining of a 12% denaturing
polyacrylamide gel showing protein molecular mass (M), total
lysates from E. coli cells transformed with PGEX-2T-HTH3
(1) and with PGEX-2T-HTH 3 (2), purified
GST-HTH3 protein (3), purified GST-HTH 3 protein
(4) is shown.
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Fig. 4.
Enzymatic activity of GST-HTH3 and
GST-HTH 3 fusion proteins: initial velocity
conditions. TH activity was measured with 25 to 100 ng of purified
fusion protein by a radioenzymatic assay using
L-[3H]tyrosine as described under
"Experimental Procedures" with various incubation times. The
activity is expressed as pmols of DOPA according to the radioactive
counts. A, pmols of DOPA increase linearly with time for
both isoforms; B, the slopes of the curves obtained in
A were plotted against enzyme quantity, showing that pmols
of DOPA/min increases linearly with the amount of enzyme.
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Fig. 5.
Lineweaver-Burk plots for GST-HTH3 and
GST-HTH 3. The enzymatic activities of
both forms were measured with a series of quantities of BH4 ranging
from 400 to 3000 µM. The inhibition by high BH4
concentrations described in Alterio et al. (10) was
observed. The curves were fitted by Kaleidagraph software
according to Dixon and Webb (18), as described under "Experimental
Procedures." micro, microgram.
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The Exon 3 Participates in the Regulation of HTH by Dopamine
and Heparin--
HTH enzymatic activity is tightly controlled in
vivo, subject to down-regulation by catecholamines as the end
products of the biosynthetic chain, and activated by phosphorylation
and polyanion binding (1).
Because retroinhibition by catecholamines is one of the major
mechanisms for controlling TH activity, we tested the effect of exon 3 skipping on the regulation of HTH activity by dopamine. Both
GST-HTH3 and GST-HTH
3 activities were analyzed with different concentrations of dopamine from 10 to 100 µM. As seen on
Fig. 6B, GST-HTH3 was more
strongly inhibited; its activity was reduced to 50% in the presence of
10 µM dopamine, whereas GST-HTH
3 was only decreased by
10%. To further characterize the kinetic parameters of the inhibition
of GST-HTH proteins by dopamine, we measured GST-HTH3 and -HTH
3
activities at various dopamine concentrations in the presence of three
different quantities of BH4 (Fig. 7). We
graphically determined the inhibition constant (Ki) of both GST-HTH isoforms for dopamine according to the Dixon method (18). The calculated Ki for dopamine was increased from 1 to 10 µM by skipping exon 3, demonstrating that
the exon 3 is implicated in the retroinhibition of TH by dopamine.

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Fig. 6.
Exon 3 contributes to the regulation of HTH
enzymatic activity. A, GST-HTH3 and GST-HTH 3 fusion
proteins were preincubated with 5 to 15 IU of heparin and then assayed.
Enzymatic activities are expressed in % of the control activity
without heparin. Variations corresponding to three independent assays
are expressed by S.E. bars. Asterisks denote a
difference from controls without heparin (*, 99% significance
according to the Fisher protected least significant difference test;
NS, non significant). B, GST-HTH3 and GST-HTH 3
fusion proteins were incubated with 10 to 100 µM dopamine
before the activity assay. Activities are shown as % of the control
activity without dopamine, and error bars are as in
A.
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Fig. 7.
Inhibition constant
(Ki) of GST-HTH3 and
GST-HTH 3 for dopamine. TH activity of
GST-HTH3 (top) and GST-HTH 3 (bottom) was
assayed in the presence of 4 different amounts of the cofactor BH4
(250, 500, 750, and 1000 µM) after preincubation with
various concentrations of dopamine (from 0 to 25 µM).
Plots were traced by expressing 1/V as a function of dopamine
concentration for the 4 different BH4 concentrations. The intersection
of the 4 curves directly gives the inhibition constant
(Ki) for dopamine, according to the Dixon method
(18). micro, microgram.
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We also studied the effect of exon 3 skipping on the activation of HTH
by heparin. GST-HTH
3 and -HTH3 activities were measured in the
presence of a series of concentrations of heparin (Fig. 6A).
GST-HTH3 activity increased gradually with heparin concentration (up to
20%), whereas no significant activation of GST-HTH
3 was observed,
strongly implicating exon 3 in the activation of HTH by heparin.
Altogether these observations provide the first demonstration that exon
3 is implicated in the HTH overall catalytic activity and, most
importantly, in the regulation of HTH activity by polyanions and by catecholamines.
 |
DISCUSSION |
A great diversity of mechanisms controlling TH activity has been
described, and among these, alternative splicing of HTH pre-mRNA is
thought to be significant (25). In the present study, we evidenced the
presence in adrenal medulla of a novel HTH isoform, HTH
3, that lacks
the third exon comprising two major phosphorylation sites,
Ser31 and Ser40. This isoform corresponds to
the translation of a previously unsuspected HTH mRNA generated by
alternative splicing recently identified in adrenal medulla and
overexpressed in adrenals of patients suffering from PSP, a
neurodegenerative disease affecting predominantly catecholaminergic
neurons of the basal ganglia (5). We also established that the exon 3 participates in the regulation of HTH enzymatic activity in
vitro.
The presence of an HTH protein isoform lacking the third exon in
vivo was evidenced by Western blotting experiments. This HTH
isoform, having the size of recombinant HTH
3, was not recognized by
an antibody specifically raised against HTH exon 3, whereas it was
clearly detected by a polyclonal antibody directed against whole rat TH
recognizing all HTH isoforms. This protein isoform was clearly detected
in PSP adrenal medulla and was about six times less abundant in control
adrenal medullas. Substantial amounts of the novel isoform were also
found in all other PSP adrenals tested (data not shown), in accordance
with the higher levels of HTH
3 mRNA previously observed in PSP
adrenals (5). This variable expression of the HTH
3 isoform indicate
that alternative splicing of HTH exon 3 may be significant. The
increased amount of HTH
3 in PSP suggests that the splicing
alteration may be a cause of the disease. This schema involving a
poorly expressed truncated enzyme isoform generated by alternative
splicing has already been proposed by Hirano et al (42). Indeed, some
familial patients with DOPA-responsive dystonia have been shown to
express a small proportion of incorrectly spliced mRNA encoding
GTP-cyclohydrolase I (the limiting enzyme in the synthesis of BH4), and
the amount correlates with the severity of the symptoms. Because
several familial cases of PSP have been described (39), we searched by
sequencing for mutations at exon 3 splice junctions within HTH gene on
genomic DNA extracted from the cerebellum of 10 PSP patients. Among
these, only three adrenal medullas were available, and they show an
expression of the HTH
3 protein (data not shown). No mutation was
found in a region of 100 bases around the donor and acceptor splice
junctions of exon 3 (data not shown), indicating that the modification
of the splicing of exon 3 is not because of a mutation of the HTH
sequence in these patients. Alternatively, there may be a modification
in the amount of splicing factors influencing the choice of splice
junctions and the inclusion/exclusion of alternative exons, such as
SF2/ASF and heterogeneous nuclear ribonucleoprotein A1 (40).
Interestingly, in Western blot experiments, an unidentified 55-kDa HTH
isoform that was not the result of alternative splicing was detected in
all tissue samples. This isoform has already been reported in previous
studies (8, 20) but was interpreted as a degradation product. However,
it may rather be a genuine HTH isoform. Indeed, its size corresponds to
(i) the use of a second AUG initiator codon located at the end of exon
1 that matches perfectly with a translation initiation sequence (21)
and/or to (ii) the result of a cleavage of HTH by calpain
(Ca2+-activated neutral protease) (22-24). The possibility
that this 55-kDa isoform is the proteolysis calpain cleavage product is of particular interest. Indeed, calpain is present in catecholaminergic tissues and activates TH in vitro (22). The 55-kDa isoform
seems to be particularly abundant in PSP adrenals (Fig. 2) but further experiments are needed to confirm its identity and any link with PSP disease.
To evaluate the role of exon 3 specifically, the enzymatic activity of
the purified fusion proteins GST-HTH3 and GST-HTH
3, which differ
only by the presence of exon 3, were compared. This work was carefully
performed in conditions in which such a comparison is meaningful, e.g.
the two fusion proteins were (i) purified in the same way and (ii)
tested precisely in initial velocity conditions with exactly the same
protocol. In these conditions, the GST-HTH proteins displayed some of
the enzymatic properties characteristic of TH, in particular inhibition
by excess BH4, heparin activation, and dopamine retroinhibition.
Moreover, the two fusion proteins displayed kinetic parameters
(Km and Ki for BH4) of the same magnitude. However,
because of the possible influence of GST on HTH activity, the kinetic parameters obtained can only be used to compare GST-HTH3 and
GST-HTH
3 proteins and thereby to evaluate the role of exon 3. Our
results show that GST-HTH
3 presents in vitro 30% of the
maximal velocity of GST-HTH3, the equivalent isoform containing exon 3. Interestingly, this lower GST-HTH
3 maximal velocity is not
associated with a modification of the Michaelis constant
(Km) for the cofactor BH4 nor of the inhibition
constant (Ki) for BH4. Thus, exon 3 may influence
the accessibility of some other components such as tyrosine and/or
ferrous iron, possibly by modifying the conformation of the enzyme. It
is unlikely that the skipping of exon 3 simply affects the catalytic
process itself, as exon 3 is not localized within the catalytic part of
the enzyme.
The third exon of HTH, absent from the novel isoform, is located in the
N-terminal regulatory part of the enzyme (26). This N-terminal region
has been implicated in the action of numerous allosteric regulatory
substances, including polyanions like heparin. Little is known about
possible direct actions of heparin on enzymatic activities within the
cell. However, it has been hypothetized that a direct interaction can
occur between intracellular proteins and heparin in the Golgi complex
or that heparin might mimic the effects of intracellular heparin-like
factors (27). Concerning TH, it has been shown that heparin reversibly
activates the enzyme probably by changing its conformation (14, 28).
Here, we show that exon 3 is involved in the activation of HTH by
heparin because GST-HTH3 is activated by heparin, whereas GST-HTH
3
is not. This result is consistent with other studies evidencing that in
rat TH, the heparin binding site is precisely localized within an exon
homologous to HTH exon 3 (15). As the interaction of the HTH N-terminal
part with heparin involves a similar mechanism as several polyanions
(1), it is probable that the absence of exon 3 also affects the
activation of HTH by other physiological polyanions such as
phospholipids, polyglutamic acid, and nucleic acids (14, 28).
Feedback inhibition of TH enzymatic activity by catecholamines is
considered as the primary mechanism by which this amine biosynthesis is
regulated (29). The regulation of TH activity by dopamine has been
extensively studied (1, 30, 31) and shown to involve a portion of the
N-terminal part of the enzyme close to Ser40 (16). Thus, we
tested if HTH exon 3 played a role in the retroinhibition process. We
found that the skipping of the HTH third exon increases the inhibition
constant (Ki) of the GST-HTH protein for dopamine by
approximately 10-fold, demonstrating that exon 3 is implicated in the
inhibition of HTH activity by dopamine. Generally, phosphorylation of
TH and retroinhibition by catecholamines act in coordination to
regulate TH activity according to the cellular requirement. For
example, phosphorylation of Ser40, mediated by most second
messenger systems, activates TH and partially relieves a tonical
inhibition by increasing the Ki of the enzyme for dopamine (32,
33). The novel HTH
3 isoform lacks Ser40, and this may
explain its escape from dopamine regulation. It also lacks
Ser31, the phosphorylation of which by two
mitogen-activated protein kinases (ERK1 and 2) has been shown to
activate TH in vitro (34, 35). However, it seems clear that
the functions carried by exon 3 are more complex than those carried by
Ser40 and Ser31 alone, because the skipping of
exon 3 (i) also abolishes activation by heparin and (ii) decreases the
activity of TH in vitro, whereas the directed mutagenesis of
Ser40 results in an activated TH (32). Thus, the HTH
3
protein, lacking such an important exon, probably exhibits particular
properties in vivo and has a genuine physiological role. The
remaining phosphorylation site for CamPKII, Ser19 (35), may
be the only site at which this isoform can be regulated.
Native TH assembles into tetramers by a stochastic process (9), and
tetramerization is required for maximal activity (36). The
N-terminal region of the protein, including exon 3, is not necessary
for the formation of the tetramer (37); thus, it is unlikely that
skipping exon 3 would prevent tetramer formation. However, if we assume
that the various HTH isoforms could be incorporated into
heterotetramers, a large number of combinations than previously described are possible, and the regulation of HTH enzymatic activity is
more complex than suspected. It would also be of importance to
establish the existence and the mechanisms of any possible dominant
effect of HTH
3 on the regulation of the enzymatic activity of the
heterotetramer. Any such dominant effect would result in the enzyme
activity being constantly maximal, in particular in PSP patients where
HTH
3 is abundant. As TH activity generates reactive free radicals
in vitro (43), such a decrease of HTH regulability could
result in oxidative stress and contribute to the degeneration of
catecholaminergic cells in these patients.
The novel HTH
3 mRNA was found in adrenal medulla but was not
detected in substantia nigra from a control (5). Possibly, the
differential expression of the HTH
3 isoform between central and
peripheral nervous system contributes to the functional diversity within the catecholaminergic system. Indeed, adrenomedullary chromaffin cells are terminally differentiated secretory cells of neural crest
origin dedicated to the synthesis, storage, and release of adrenaline
and noradrenaline and are phenotypically and functionally distinct from
catecholaminergic neurons of the central nervous system (38). Another
argument for the possible link between alternative splicing of TH
premessenger RNA and phenotypic diversity within the catecholaminergic
system is the increased amount of HTH isoforms containing exon 2 (HTH3
and 4) in the adrenal medulla compared with the central nervous system
(4). This is also probably significant because the isoform 3 has
recently been shown to present specific regulatory properties after
cAMP-dependant protein kinase phosphorylation (10).
It is clear that exon 3 plays a role in the regulation of HTH enzymatic
activity. Interestingly, this part of TH may also have a role in other
species. Indeed, the region homologous to the HTH exon 3 is also
alternatively spliced in rat (17) and in Drosophila (41)
(Fig. 8). Moreover, in
Drosophila, the TH isoform that differs in the region
corresponding to exon 3 is specifically expressed in the hypoderm and
has been proposed to be hyperactive and contribute to the synthesis of
the large amounts of dopamine within the cuticle.

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|
Fig. 8.
Comparative alternative splicing of
Drosophila TH (DTH), HTH, and RTH
premessenger RNA. Exons are represented by boxes, which
are filled if alternatively spliced. The homologous regions of HTH exon
3 in RTH and DTH are indicated by a line. The
asterisk represents the major site of phosphorylation of TH
(Ser40).
|
|
Finally, the identification of a variably expressed HTH isoform that
lacks an exon implicated in enzymatic activity regulation supports the
view that HTH alternative splicing may be involved in some neurological
diseases and/or in the functional diversity of the catecholaminergic system.
 |
Ackowledgements |
We are grateful to Dr. J. F. Reinhard
for providing the anti-TH antibody. Thanks to O. Corti for providing
the HTH1-transfected cell line. Special thanks to V. Berthelier for
technical advice and helpful discussions.
 |
FOOTNOTES |
*
This work was made possible by financial support from the
CNRS, Ministère de l'Enseignement Supérieur et de la
Recherche, Rhône-Poulenc-Rorer and the EEC Science Progromme.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a fellowship from the Institut de Recherche sur la
Moelle Epiniere.
To whom correspondence should be addressed: LGN,
Bâtiment CERVI étage 5, Hôpital de la
Pitié-Salpêtrière, 83 Bd de l'Hôpital, 75013 Paris, France. Tel.: 33 1 4217 7559; Fax: 33 1 4217 7533; E-mail: sdumas{at}infobiogen.fr.
The abbreviations used are:
TH, tyrosine
hydroxylase; PSP, progressive supranuclear palsy; GST, glutathione
S-transferase; RTH, rat TH; HTH, human TH.
 |
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.