species differences in the expression of multiple tyrosine hydroxylase protein isoforms

Species differences in the expression of multiple tyrosine

hydroxylase protein isoforms

John W. Haycock

Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana,

USA

Abstract

In subprimates, a single form of tyrosine hydroxylase (TH) is

expressed, whereas two TH protein isoforms have been

identified in monkeys and four isoforms have been demon-

strated in humans. In order to establish the evolutionary

pattern/emergence of these multiple TH isoforms, adrenal

medullae from different mammalian species were analyzed by

blot immunolabeling using pan-specific TH antibodies and

antibodies specific to each of the four human TH isoforms.

The expression of multiple TH isoforms was primate specific

and restricted to anthropoids: only a single TH isoform was

detected in adrenal medullae from several subprimate and

prosimian species (six species from four families), while two

TH isoforms were found in all of the anthropoid species

studied. The presence of four TH isoforms could only be

demonstrated in human specimens. Contrary to previous

suggestions, only one TH protein isoform was found in rats

and only four TH protein isoforms were found in humans.

Keywords: alternative splicing, catecholamine, human,

mammal, phylogeny, primate.

J. Neurochem. (2002) 81, 947–953.

Tyrosine hydroxylase [EC 1.14.16.2; tyrosine 3-monooxy-

genase; L-tyrosine tetrahydropteridine: oxygen oxidoreductase

(3-hydroxylating); TH], which catalyzes the first, rate-

limiting step in catecholamine biosynthesis, is a tetrameric

protein (molecular weight �240 000) encoded by a single

gene in all species studied to date (cf. Kumer and Vrana

1996; Fitzpatrick 1999). In subprimates, the holoenzyme is

thought to be an oligomer composed of four identical

subunits (Goodwill et al. 1997), synthesized from a single

form of mRNA encoding TH (Brown et al. 1987; Ichikawa

et al. 1990). In humans, however, two alternative splicing

events produce four different forms of TH mRNA that

encode four different protein isoforms (Grima et al. 1987;

Kaneda et al. 1987; Kobayashi et al. 1987; O’Malley et al.

1987; Coker et al. 1990), and the presence of all four of the

cognate protein isoforms has been demonstrated in human

brain and adrenal medulla (Haycock 1991; Lewis et al.

1993). By contrast, only two TH mRNAs encoding

different TH isoforms are found in Old World (Macaca)

and New World (Callithrix) monkeys (Ichikawa et al. 1990;

Ichinose et al. 1993), and only the two predicted TH

protein isoforms are found in macaque brain and adrenal

medulla (Lewis et al. 1994). Consistent with these data, the

genomic sequence of macaque TH DNA was found to be

incompatible with more than a single alternative splicing

event. In the same analyses (Ichinose et al. 1993), however,

the genomic sequences of gibbon, orangutan, gorilla, and

chimpanzee TH DNA were found to be compatible with

two alternative splicing events, allowing for the potential

existence of four different TH mRNAs and cognate protein

isoforms in these species.

The single TH protein/mRNA isoform in subprimates

versus two in monkeys raises the question of when in

evolution an additional TH isoform arose. And, the potential

additional alternative splicing event in higher primates raises

the question of whether the expression of four TH protein

isoforms is human specific. To address these questions and

the possible existence of yet additional, novel, TH protein

isoforms both in rats (Schussler et al. 1995; Laniece et al.

1996) and in humans (Dumas et al. 1996), the present study

employed blot immunolabeling techniques with pan-specific

Resubmitted manuscript received January 23, 2002; accepted February

8, 2002.

Address correspondence and reprint requests to John W. Haycock,

LSUHSC-BIOCHEM, 1100 Florida Avenue, New Orleans, LA 70119,

USA. E-mail: [email protected]

Abbreviations used: PAGE, polyacrylamide gel electrophoresis;

PC, pheochromocytoma; SDS, sodium dodecyl sulfate; TH, tyrosine

hydroxylase.

Journal of Neurochemistry, 2002, 81, 947–953

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 81, 947–953 947

TH antibodies and antibodies specific to each of the four

human TH isoforms to analyze the TH protein isoforms

present in catecholaminergic tissues from a wide range of

mammalian species.

Materials and methods

Sample preparation

Adrenal medullae were dissected free-hand (after warming to ) 10

to ) 20�C in a cryostat) from adrenal glands that had been removed

and stored frozen () 80�C) after the animals had been killed or

autopsied. Portions of the adrenal and pheochromocytoma samples

were solubilized in a sodium dodecyl sulfate (SDS)-containing

solution by sonication and heating, cleared by centrifugation, and

prepared for SDS–polyacrylamide gel electrophoresis (PAGE), as

previously described (Haycock 1991). Human or rat liver extract

was added to the lanes, as necessary, to achieve at least 20 lg of

total protein per lane. TH standards were as previously described

(Haycock 1993b).

Blot immunolabeling

As described elsewhere in detail (Haycock 1993a; Ordway et al.

1994), aliquots were subjected to SDS–PAGE (9% slab gels), and

the proteins in the separating gel were transferred electrophoret-

ically to nitrocellulose sheets. After protein staining (Ponceau S)

had been documented xerographically, the transfers were then

destained/quenched in blot buffer [Dulbecco’s phosphate-buffered

saline (Gibco, Rockville, MD, USA), 10 mM Tris-HCl (pH 7.6),

0.05% (w/v) Tween 20, 0.01% sodium azide] containing 1% (w/v)

polyvinylpyrrolidone (Haycock 1993a). The transfers were then

sequentially incubated (1 h at room temperature) with the primary

antibody, the secondary antibody (0.8–1.0 lg/mL), and either

[125I]protein A (200 kcpm/mL; NEN, Boston, MA, USA) in blot

buffer containing polyvinylpyrrolidone or enhanced chemilumines-

cence reagents (Amersham, Piscataway, NJ, USA). The transfers

were rinsed five times (2 · 2 min, 3 · 5 min) with blot buffer

after incubation with each of the reagents. The secondary

antibodies used were affinity-purified swine anti-rabbit Ig (0.8 lgIgG/mL; Dako, Carpinteria, CA, USA) and affinity-purified rabbit

anti-mouse IgG1 or peroxidase-rabbit anti-mouse IgG1 (1 lg IgG/mL;

Dako). Immunoreactivity was visualized autoradiographically

(XAR film; Kodak, Rochester, NY, USA) and quantitated by

gamma counting of excised bands and blanks. Relative abundances

of TH isoforms were calculated by interpolation from the dynamic

working ranges of TH standard curves run on the same blot

(Haycock 1993b).

Antibodies

The pan-specific anti-TH antibodies (which have been shown to

recognize essentially all mammalian TH isoforms) used were

affinity-purified rabbit polyclonal and mouse monoclonal antibodies

raised against SDS-denatured, native rat TH (Haycock 1989,

1993b). These antibodies react equally with the human TH isoforms

(Haycock 1993b). Affinity-purified rabbit anti-peptide antibodies

specific for each of the four different human TH isoforms were as

described in detail previously (Haycock 1991, 1993b; Lewis et al.

1993).

Results and discussion

Emergence of multiple TH isoforms in anthropoids

TH is present as a prominent 58–64-kDa band in the adrenal

medullae of all mammalian species tested, as illustrated by

the blots in the upper and lower panels of Fig. 1, which show

immunoreactivities with the polyclonal and monoclonal anti-

pan TH antibodies. In the upper middle panel, blot immuno-

labeling of the same samples with antibodies to type 1 human

TH (HTH1) demonstrates (1) the lack of immunoreactivity of

rodent TH with antibodies to HTH1 and (2) the lack of cross-

reactivity of this antibody with type 2 human TH (HTH2).

The lack of type 1 TH immunoreactivity in rodent TH

reflects the substitution of Val Thr (VT) for Ile Met (IM)

residues immediately N-terminal to Ser31 which, of all

mammalian TH cloned to date, occurs only in rodent TH

Fig. 1 Blot immunolabeling of tyrosine hydroxylase (TH) in primate

and subprimate species. Replicate gels were loaded with aliquots of

adrenal medulla extracts and samples from the indicated sources and

subjected to sodium dodecyl sulfate–polyacrylamide gel electrophor-

esis (SDS–PAGE). After immunolabeling of sections (�40–80 kDa) of

the blots with the indicated antibodies, immunoreactivity was devel-

oped ([125I]protein A for rabbit antibodies and enhanced chemilumi-

nescence for mouse antibodies) as described under Materials and

methods. The immunogen for each antibody is given in parentheses.

[In a separate experiment, alligator TH was detected by the rabbit but

not the mouse anti-rat TH as a single �45-kDa band (data not

shown).]

948 J. W. Haycock

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 81, 947–953

(Grima et al. 1985; Ichikawa et al. 1991). By contrast,

guinea pig TH does react with the rabbit anti-HTH1, arguing

against the contention that Cavia species are members of the

rodent family (cf. Martignetti and Brosius 1993). The lower

middle panel demonstrates (1) that HTH2 was not detected in

subprimate species, (2) that HTH2 was detected in anthro-

poid but not prosimian primates, and (3) the lack of cross-

reactivity of the HTH2 antibody with HTH1. As indicated in

Table 1, which lists the primate species analyzed in the

present experiments, a total of six different prosimian species

from four different families was studied. As shown for the

Galago sample shown in Fig. 1, HTH1 but not HTH2 was

detected in each of the prosimian samples (data not shown).

Thus, the presence of multiple TH isoforms was restricted to

the anthropoid branch of primate species.

Mallet and coworkers have identified a second TH mRNA

species in rat adrenal and pheochromocytoma (PC) cells,

created by using an alternative donor site in exon 2, that

comprises 5% of the total TH mRNA in rat adrenal medulla

(Schussler et al. 1995; Laniece et al. 1996), which is

comparable with the combined relative abundances of

HTH3 plus HTH4 mRNA in human adrenal (Coker et al.

1990; see also below). The cognate TH protein isoform

would be missing the last 33 amino acid residues encoded

by exon 2 and, hence, migrate as a �3.6-kDa lower band in

SDS–PAGE. Using a polyclonal antiserum to TH, Schussler

et al. (1995) reported the presence of several TH-immuno-

reactive bands in samples from rat adrenal gland. However,

the only lower molecular weight species observed in Fig. 1

with the affinity-purified rabbit anti-rat TH were in lanes

containing Galago and sheep adrenal samples. Moreover,

these lower molecular weight bands were not detected by

mouse anti-rat TH, whose epitope resides very near the

N-terminus (i.e. within the residues encoded by exon 1)

(Haycock 1991, 1993b), indicating that these lower molecu-

lar weight bands were produced by N-terminal proteolysis as

opposed to the proposed alternative splice variant, which

would have been detected by the mouse anti-rat TH. In fact,

blot immunolabeling of over a 50-fold range of rat adrenal

medullary (Fig. 2) or PC12 cell (data not shown) loads with

mouse anti-rat TH failed to reveal the presence of additional

TH bands. Thus, the existence of multiple TH mRNA

species does not necessarily translate directly into the

existence of their cognate protein isoforms. Based upon

the sensitivity of the present assays, should the cognate TH

protein isoform be expressed in either rat adrenal or PC12

cells, its relative abundance would have to be less than 1%

of full-length TH. In any case, while an mRNA template is

required for translation, the rate of translation and/or the

turnover of the translated protein are indeterminate. Given

that isoform-specific anti-peptide antibodies can be gener-

ated, the existence and quantitation of such postulated

Table 1 Primate speciesa

Family Common name Genus Species n

Prosimii

Lemeridae Lemur Lemur coronatus 1

Lemur Microcebus murinus 1

Indriidae Sifaka Propithecus verreauxi 1

Lorisidae Loris Nycticebus coucang 1

Bushbaby Galago crassicaudatus 1

Tarsiidae Tarsier Tarsius syrichta 1

Anthropoidae

Cebidae Squirrel monkey Saimiri boliviensis 3

Callithricidae Marmoset Callithrix jacchus 2

Tamarin Leontocebus imperator 1

Tamarin Saquinis oedipus 2

Cercopithecidae Rhesus monkey Macaca mulatta 5

Cynomolgous monkey Macaca irus 2

Green monkey Cercopithicus aethiops 1

Yellow baboon Papio cynocephalus 1

Yellow/anubis baboon Papio [mix] 2

Pongidae White-handed gibbon Hylobates lar 1

Orangutan Pongo pygmaeus 1

Gorilla Gorilla gorilla 1

Chimpanzee Pan troglodytes 13

Hominidae Human Homo sapiens 15

aTaxonomical nomenclature taken from Morris (1965). n, number of subjects.

Multiple tyrosine hydroxylase isoforms 949


splicing variant proteins requires specific immunological

determination.

Multiple TH isoforms in anthropoids

Figure 3 presents a sampling of the blot immunolabeling

results obtained with adrenal medullary samples from various

families/species within the anthropoid suborder. As indicated

in the upper and lower panels, approximately equal amounts

of total TH were analyzed. HTH1 and HTH2 were both

present in all anthropoid samples analyzed. However, the

relative abundance of HTH2 was distinctly lower in each of

the New World monkey samples (cebids and callithricids; cf.

Table 1 and Fig. 3). Quantitation of HTH1 and HTH2 levels

indicated that, as previously reported for rhesus monkeys and

humans (Lewis et al. 1993, 1994), both isoforms were

present in approximately equal abundance in Old World

monkeys (cercopithecids), gibbons/great apes (pongids), and

humans, whereas HTH2 comprised only 15–20% of total TH

in New World monkeys. Although an intermediate value was

obtained for the Cercopithecus sample, confirmation of this

would require analysis of additional samples. HTH3 and

HTH4, however, were detected only in human samples, as

described in detail previously (Haycock 1991, 1993b; Lewis

et al. 1993).

What physiological importance the multiple TH protein

isoforms may play must first be considered in light of both

their relative abundances and their distributions within the

nervous system. For example, based on protein data, there is

no evidence for the existence of HTH1 only or HTH2 only

cells, and the association of TH subunits into the holoenzyme

appears to be a stochastic process (Lewis et al. 1993, 1994).

Thus, given that types 1–4 all possess relatively similar

catalytic activities, the presence of small proportions of

HTH3 and HTH4 is unlikely to create discernible differences

in the overall activity/reactivity of the entire TH population.

On the other hand, HTH2 comprises a substantial proportion

of total TH in higher primates. Grima et al. (1987) initially

suggested that the additional four amino acids immediately

upstream of Ser31 in HTH2, created a new phosphorylation

site for calcium/calmodulin-dependent protein kinase II.

Subsequently, Ser31 in the type 1 isoform was identified as a

substrate for extracellular signal-regulated protein kinases 1

and 2 (Haycock et al. 1992), allowing for the possibility not

that a new site was created but that the protein kinase

specificity for the site could be different. However, as

indicated by the analysis of 32P incorporation in situ, the

phosphorylation of Ser31 in HTH2 was not regulated by

Fig. 3 Blot immunolabeling of tyrosine hydroxylase (TH) in primates.

Replicate gels were loaded with aliquots of representative adrenal

medulla extracts and samples from the indicated sources and sub-

jected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS–PAGE). After blot immunolabeling with the indicated antibodies,

immunoreactivity was developed as described under Materials and

methods. Ra, rabbit anti; Ma, mouse anti; BSA, bovine serum albumin;

CAT, catalase. (Note: the exposure time of the HTH3 + HTH4 blot was

�4-fold longer than those of the other blots.)

Fig. 2 Blot immunolabeling of tyrosine hydroxylase (TH) in rat

adrenal. Aliquots of pooled rat adrenal extracts containing the

indicated amounts of protein were subjected to sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blot

immunolabeling of �40–80-kDa blot sections with mouse anti-rat TH,

secondary antibody and [125I]protein A. Immunoreactivity was visual-

ized by autoradiography.

950 J. W. Haycock


either of these protein kinases (Haycock 1993c). In that

Ser31 phosphorylation appears to be involved in depolariza-

tion-dependent activation of TH, both in chromaffin cells

(Salvatore et al. 2001) and in brain (Lindgren et al. 2002),

one could speculate that the additional TH isoforms in higher

primates serve to decrease its activation in response to

depolarization and/or activators of the extracellular signal-

regulated protein kinases 1 and 2, thereby decreasing the

potential for cytotoxicity associated with the products of

tyrosine hydroxylation.

As mentioned in the introduction, Ichinose et al. (1993)

found that, in addition to human sequences, the genomic

sequences of gibbon, orangutan, gorilla, and chimpanzee TH

DNA were also compatible with two alternative splicing

events, allowing for the potential existence of four different

TH mRNAs and cognate protein isoforms in these species.

Although limited numbers of samples were available from

species other than humans and chimpanzees, no evidence for

more than two TH protein isoforms was found in any of the

pongid samples, all of which were analyzed extensively.

Although the human adrenals in the present study were all

from adults (20–64 years old), the chimpanzee adrenals

came from animals ranging in age from neonate to 21 years

old. There were no discernible age- or sex-related differ-

ences in the relative abundances of human TH protein

isoforms in either cohort (data not shown). Also, in a

systematic study of post-natal development and aging in

autopsied human striatum, the relative abundances of the

four human TH isoforms were invariant (Haycock et al.

1997).

Based on RNase protection assays in samples of human

adrenal medullae, Dumas et al. (1996) identified three

additional mRNA splice variants of human TH produced

by the elimination of exon 3. The resulting human TH

isoforms would lack the 75 amino acids encoded by exon 3

and thus migrate in SDS–PAGE as bands approximately

8 kDa lower than their cognate isoforms containing exon 3.

Whereas in normal adrenal medullae the relative abundance

of these splice variants was 4–6% of total human TH mRNA

(comparable with that of HTH3 and HTH4 mRNA; Coker

et al. 1990), in adrenal medullae from subjects who suffered

from progressive supranuclear palsy, these exon 3-less

variants comprised up to 34% of total human TH mRNA.

Given the potential importance of these observations,

numerous human adrenal medullae and pheochromocytoma

were analyzed to determine whether proteins corresponding

to the predicted translation products could be found.

However, with the possible exception of the progressive

supranuclear palsy adrenal medulla sample in lane 3 in which

very faint lower molecular weight immunoreactivity was

present, the profiles of immunoreactivity with mouse anti-rat

TH (which would cross-react with all of the human TH

protein isoforms proposed to date) revealed only the

prominent HTH1 + 2 band and the upper, lower abundance

HTH3 + 4 band (Fig. 4).

The present results emphasize the importance of testing

the essential assumptions that are involved when making

inferences for proteins from mRNA data. For example, as

in the rat adrenal gland (Schussler et al. 1995), whereas

Bodeau-Pean et al. (1999) detected numerous immuno-

reactive bands in human adrenals with a polyclonal anti-

pan TH antiserum, including a lower molecular weight

band not recognized by an anti-TH exon 3 antibody, the

possibility that this band was an N-terminal proteolytic

product was not ruled out and antibodies that would

positively recognize any of the imputed TH isoforms were

not tested.

The RNase assays used to identify the apparently novel

human TH variants are not without limitations. For

example, an alternative approach using RT–PCR with the

Fig. 4 Blot immunolabeling of tyrosine hydroxylase (TH) in human

pheochromocytomas and adrenal medullae. Aliquots of extracts from

human pheochromocytoma samples (n ¼ 6) and human adrenal

medullae samples (n ¼ 9) containing similar amounts of total TH were

subjected to sodium dodecyl sulfate–polyacrylamide gel electrophor-

esis (SDS–PAGE) and blot immunolabeling with mouse anti-rat TH,

secondary antibody and [125I]protein A. Immunoreactivity was visual-

ized by autoradiography. Because a long exposure was used to reveal

potential lower molecular weight bands, the upper, less intense

HTH3 + HTH4 band was relatively obscured in most lanes by the

predominant HTH1 + HTH2 band. PSP, post-mortem samples taken

from three subjects diagnosed with progressive supranuclear palsy.



pheochromocytoma samples shown in Fig. 4 failed to

confirm the existence of any of the variants reported by

Dumas et al. (1996) (K. O’Malley, personal communica-

tion). Nonetheless, whether or not alternatively spliced

mRNA species can be detected, the critical issue is whether

or not the cognate protein species exists. As such, certain

limitations of blot immunolabeling assays must also be

considered. As noted above, difficulties with blot immuno-

labeling studies can arise from cross-reactivities with other

antigens in crude antisera and from the presence of multiple/

undetermined epitopes on the antigen with which a specific

antibody can react. However, depending upon the immuno-

gen selected, polyclonal anti-peptide antibodies can be

targeted to react specifically and selectively with a specific

antigen at a specific locus and, by definition, monoclonal

antibodies react with a specific epitope. As such, these assays

can be gainfully employed as positive identifiers of protein

species, evidenced by the isoform-specific anti-peptide

antibodies and the monoclonal mouse antibody against an

epitope in the extreme N-terminus of TH used in the present

studies. And, given the numerous �sandwich�/detectionmethods currently available, determining relative abundances

is essentially limited only by the separation of isoforms in

SDS–PAGE. To this point it should be added that the

immunoreactivities of HTH1 and HTH2 – which differ by

only four amino acid residues – can be distinguished as two

separate bands in the present system if the gels are run the

full length (�5.5 cm) and chromogenic detection of peroxi-

dase-labeled secondary antibody is used (J. W. Haycock,

unpublished observations).

Acknowledgements

The primate specimens used in these studies were acquired through

the National Neurological Research Bank (Los Angeles), the

Cooperative Human Tissue Network (University of Alabama,

Birmingham), Duke University Primate Center, Yerkes Regional

Primate Research Center, University of Pittsburgh Primate Center,

University of South Alabama Primate Center, Marmoset Research

Center Oak Ridge, New Iberia Research Center, LEMSIP (NYU

Medical Center), and Buckshire Corporation. Primate tissue

acquisitions were facilitated by the Primate Supply Information

Clearinghouse (University of Washington). Subprimate specimens

were obtained from PelFreez Incorporated. This research was

funded by USPHS grants MH00967, MH55208, and NS25134.

References

Bodeau-Pean S., Ravassard P., Neuner-Jehle M., Faucheux B., Mallet J.

and Dumas S. (1999) A human tyrosine hydroxylase isoform

associated with progressive supranuclear palsy shows altered

enzymatic activity. J. Biol. Chem. 274, 3469–3475.

Brown E. R., Coker G. T. III and O’Malley K. L. (1987) Organization

and evolution of the rat tyrosine hydroxylase gene. Biochemistry

26, 5208–5212.

Coker G. T. III, Studelska D., Harmon S., Burke W. and O’Malley K. L.

(1990) Analysis of tyrosine hydroxylase and insulin transcripts in

human neuroendocrine tissues. Mol. Brain Res. 8, 93–98.

Dumas S., Le Hir H., Bodeau-Pean S., Hirsch E., Thermes C. andMallet J.

(1996) New species of human tyrosine hydroxylase mRNA are pro-

duced in variable amounts in adrenal medulla and are overexpressed

in progressive supranuclear palsy. J. Neurochem. 67, 19–25.

Fitzpatrick P. F. (1999) Tetrahydropterin-dependent amino acid hydroxy-

lases. Annu. Rev. Biochem. 68, 355–381.

Goodwill K. E., Sabatier C., Marks C., Raag R., Fitzpatrick P. F. and

Stevens R. C. (1997) Crystal structure of tyrosine hydroxylase at

2.3 A and its implications for inherited neurodegenerative diseases.

Nat. Struct. Biol. 4, 578–585.

Grima B., Lamouroux A., Blanot F., Faucon Biguet N. and Mallet J.

(1985) Complete coding sequence of rat tyrosine hydroxylase

mRNA. Proc. Natl Acad. Sci. USA 82, 617–621.

Grima B., Lamouroux A., Boni C., Julien J.-F., Javoy-Agid F. and Mallet

J. (1987) A single human gene encoding multiple tyrosine

hydroxylases with different predicted functional characteristics.

Nature 326, 707–711.

Haycock J. W. (1989) Quantitation of tyrosine hydroxylase protein

levels: spot immunolabeling with an affinity-purified antibody.

Anal. Biochem. 181, 259–266.

Haycock J. W. (1991) Four forms of tyrosine hydroxylase are present in

human adrenal medulla. J. Neurochem. 56, 2139–2142.

Haycock J. W. (1993a) Polyvinylpyrrolidone as a blocking agent in

immunochemical studies. Anal. Biochem. 208, 397–399.

Haycock J. W. (1993b) Multiple forms of tyrosine hydroxylase in human

neuroblastoma cells: quantitation with isoform-specific antibodies.

J. Neurochem. 60, 493–502.

Haycock J. W. (1993c) Phosphorylation of tyrosine hydroxylase iso-

forms in human neuroblastoma cells. Mol. Biol. Cell 41, 406a.

Haycock J. W., Ahn N. G., Cobb M. H. and Krebs E. G. (1992) ERK1

and ERK2, two microtubule-associated protein kinases, mediate

the phosphorylation of tyrosine hydroxylase at serine 31 in situ.

Proc. Natl Acad. Sci. USA 89, 2365–2369.

Haycock J. W., Zhong X.-H., Hornykiewicz O., Furukawa Y. and Kish

S. J. (1997) Presynaptic dopaminergic markers in human striatum

during development and aging. Soc. Neurosci. Abstract. 23, 41.

Ichikawa S., Ichinose H. and Nagatsu T. (1990) Multiple mRNAs of

monkey tyrosine hydroxylase. Biochem. Biophys. Res. Commun.

173, 1331–1336.

Ichikawa S., Sasaoka T. and Nagatsu T. (1991) Primary structure of

mouse tyrosine hydroxylase deduced from its cDNA. Biochem.

Biophys. Res. Commun. 176, 1610–1616.

Ichinose H., Ohye T., Fujita K., Yoshida M., Ueda S. and Nagatsu T.

(1993) Increased heterogeneity of tyrosine hydroxylase in humans.

Biochem. Biophys. Res. Commun. 195, 158–165.

Kaneda N., Kobayashi K., Ichinose H., Kishi F., Nakazawa A., Kuro-

sawa Y., Fujita K. and Nagatsu T. (1987) Isolation of a novel

cDNA clone for human tyrosine hydroxylase: alternative RNA

splicing produces four kinds of mRNA from a single gene. Bio-

chem. Biophys. Res. Commun. 146, 971–975.

Kobayashi K., Kaneda N., Ichinose H., Kishi F., Nakazawa A., Kuro-

sawa Y., Fujita K. and Nagatsu T. (1987) Isolation of a full length

cDNA clone encoding human tyrosine hydroxylase type 3. Nucleic

Acids Res. 15, 6733.

Kumer S. C. and Vrana K. E. (1996) Intricate regulation of tyrosine hy-

droxylase activity and gene expression. J. Neurochem. 67, 443–462.

Laniece P., Le Hir H., Bodeau-Pean S., Charon Y., Valentin L., Thermes

C., Mallet J. and Dumas S. (1996) A novel rat tyrosine hydroxylase

mRNA species generated by alternative splicing. J. Neurochem.

66, 1819–1825.

952 J. W. Haycock


Lewis D. A., Melchitzky D. S. and Haycock J. W. (1993) Four isoforms

of tyrosine hydroxylase are expressed in human brain. Neuro-

science 54, 477–492.

Lewis D. A., Melchitzky D. S. and Haycock J. W. (1994) Expression and

distribution of two isoforms of tyrosine hydroxylase in macaque

monkey brain. Brain Res. 656, 1–13.

Lindgren N., Goiny M., Herrera-Marschitz M., Haycock J. W., Hokfelt

T. and Fisone G. (2002) Activation of extracellular signal-regulated

kinases 1 and 2 by depolarization stimulates tyrosine hydroxylase

phosphorylation and dopamine synthesis in rat brain. Eur.

J. Neurosci. 15, 769–773.

Martignetti J. A. and Brosius J. (1993) Neural BC1 RNA as an evolu-

tionary marker: guinea pig remains a rodent. Proc. Natl Acad. Sci.

USA 90, 9698–9702.

Morris D. (1965) The Mammals: a Guide to the Living Species, pp. 105–

162. Harper & Row, New York.

O’Malley K. L., Anhalt M. J., Martin B. M., Kelsoe J. R., Winfield S. L.

and Ginns E. I. (1987) Isolation and characterization of the human

tyrosine hydroxylase gene: identification of 5¢-alternative splice

sites responsible for multiple mRNAs. Biochemistry 26, 6910–

6914.

Ordway G. A., Smith K. S. and Haycock J. W. (1994) Elevated tyrosine

hydroxylase in the locus coeruleus of suicide victims. J. Neuro-

chem. 62, 680–685.

Salvatore M. F., Waymire J. C. and Haycock J. W. (2001) Depolariza-

tion-stimulated catecholamine biosynthesis: involvement of protein

kinases and tyrosine hydroxylase phosphorylation sites in situ.

J. Neurochem. 79, 349–360.

Schussler N., Boularand S., Li J. Y., Peillon F., Mallet J. and Biguet N. F.

(1995) Multiple tyrosine hydroxylase transcripts and immuno-

reactive forms in the rat: differential expression in the anterior

pituitary and adrenal gland. J. Neurosci. Res. 42, 846–854.



species differences in the expression of multiple tyrosine hydroxylase protein isoforms

Documents