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 immunolabeling 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 (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 anthropoid 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.
Figure 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 electrophoresis (SDS–PAGE). After immunolabeling of sections (∼40–80 kDa) of the blots with the indicated antibodies, immunoreactivity was developed ([125I]protein A for rabbit antibodies and enhanced chemiluminescence 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).]
Download figure to PowerPoint
Table 1. Primate species a
| Cebidae||Squirrel monkey||Saimiri||boliviensis||3|
| Cercopithecidae||Rhesus monkey||Macaca||mulatta||5|
| Pongidae||White-handed gibbon||Hylobates||lar||1|
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-immunoreactive 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 molecular 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 generated, the existence and quantitation of such postulated splicing variant proteins requires specific immunological determination.
Figure 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 visualized by autoradiography.
Download figure to PowerPoint
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).
Figure 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 subjected 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. Rα, rabbit anti; Mα, 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.)
Download figure to PowerPoint
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 either of these protein kinases (Haycock 1993c). In that Ser31 phosphorylation appears to be involved in depolarization-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 differences 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).
Figure 4. Blot immunolabeling of tyrosine hydroxylase (TH) in human pheochromocytomas and adrenal medullae. Aliquots of extracts from human pheochromocytoma samples (n = 6) and human adrenal medullae samples (n = 9) containing similar amounts of total TH were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and blot immunolabeling with mouse anti-rat TH, secondary antibody and [125I]protein A. Immunoreactivity was visualized 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.
Download figure to PowerPoint
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 immunoreactive 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 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 communication). 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 immunolabeling 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 immunogen 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’/detection methods 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 peroxidase-labeled secondary antibody is used (J. W. Haycock, unpublished observations).