©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Human Dopamine -Hydroxylase in Mammalian Cells Infected by Recombinant Vaccinia Virus
MECHANISMS FOR MEMBRANE ATTACHMENT (*)

Lela Houhou , Annie Lamouroux , Nicole Faucon Biguet , Jacques Mallet (§)

From the (1) Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, CNRS, 91198 Gif-sur-Yvette, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Dopamine -hydroxylase (DBH) is found in neurosecretory vesicles in both membrane-bound and soluble forms. We expressed various human DBH cDNAs in two mammalian cell lines, using the vaccinia virus expression system. The expression of a full-length DBH cDNA (DBH-f) reproduced the native DBH electrophoretic pattern and led to the synthesis of an active enzyme composed of two subunits of 77 and 73 kDa. In contrast, a truncated cDNA lacking the first ATG (DBH-t) generated a single band of 73 kDa. Analysis of mutated recombinant clones demonstrates that the two polypeptides do not result from the use of an alternative translation initiator codon. These results, combined with deglycosylation experiments, allow us to attribute the double band pattern to an optional cleavage of the signal peptide. When the NH-terminal extremity is shortened, cleavage becomes obligatory, underlining the role of the first 14 amino acids in the regulation of the cleavage of the signal peptide. Subcellular analysis of recombinant DBH-t and DBH-f proteins indicates that DBH is anchored to the membrane by two distinct mechanisms; one of them is due to the non-removal of the signal peptide, whereas the second one is independent of the presence of the signal sequence. Moreover, quantification of the fractionation experiments suggests that the two modes of membrane attachment are additive.


INTRODUCTION

Dopamine -hydroxylase (DBH)() (EC 1.14.17.1) catalyzes the conversion of dopamine to norepinephrine, a step in the biosynthetic pathway of catecholamine neurotransmitters (1). The enzyme is found inside the chromaffin granules of the adrenal medullary cells (2) and in the neurosecretory vesicles of the noradrenergic neurons of the peripheral and central nervous systems (3, 4) . The biochemical properties of DBH are well established, mostly with the bovine enzyme (for a review, see Ref. 5). DBH is a copper-containing glycoprotein. The enzyme is known to exist as both a soluble and a membrane-bound protein in similar amounts according to activity measurements (6) . Both forms are tetramers composed of two disulfide-linked dimers, with the dimers held together by noncovalent interactions (7) .

During the last 10 years, efforts have been made to understand the structure of the soluble and membrane-bound form of DBH and to elucidate the mechanism by which the enzyme is anchored to the membrane. The amino acid sequences deduced from the cDNAs coding for human (8, 9) , bovine (10, 11, 12, 13, 14) , and rat (15) DBH are highly similar and reveal the presence of an amino-terminal signal peptide as the only hydrophobic domain of the protein. Expression of a full-length bovine DBH cDNA in rat pheochromocytoma PC12 cells demonstrated that the two forms are encoded by the same mRNA (16) . The structural differences between the soluble and the membrane-bound DBH are not entirely understood. It is assumed that the soluble form is composed of a single 73-kDa subunit as analyzed by SDS-polyacrylamide gel electrophoresis, whereas the membrane-bound form exhibits, in addition to the latter band, a 77-kDa subunit. The comparison of the amino-terminal sequence of the purified soluble form of bovine DBH (17, 18) with that deduced from the cDNA clone establishes that in the 73-kDa subunit the signal peptide is always cleaved. In contrast, the amino-terminal sequence of membraneous DBH indicates that 20-30% of the molecules, depending on the authors, have retained the signal peptide (10, 19) . More precisely, most of the 77-kDa subunits but none of the 73-kDa subunits of membraneous DBH contain an uncleaved signal sequence (10) . All of these results suggest that the signal peptide is not systematically cleaved and may thereby anchor the enzyme to the membrane. The current model proposes that soluble DBH is a homotetramer of the 73-kDa processed subunit, whereas membraneous DBH would be a heterotetramer of the 73- and 77-kDa subunits. However, some data indicate a possibly more complex situation. There are controversial reports of the 77-kDa subunit in the purified soluble bovine DBH, as analyzed by SDS-polyacrylamide gel electrophoresis (16, 18, 19, 20) . Additionally, deglycosylation of both forms of purified bovine DBH resulted in a single band of lower molecular weight (21) , although this finding was not confirmed by Bon et al.(22) . More importantly, the expression in Drosophila Schneider 2 cells of a bovine DBH cDNA lacking its own signal sequence led to the synthesis of both soluble and membraneous forms (23) . This latter result demonstrates that DBH can be membrane-bound independently of the presence of the signal peptide.

At present, several features of DBH expression remain unresolved. Mainly, the structural differences between the 73- and the 77-kDa subunits are unclear. According to the above mentioned reports, this size heterogeneity may result from alternative translation initiation, differential carbohydrate composition, or partial cleavage of the signal peptide. Moreover, the correlation between the two subunits and the soluble and membrane-bound forms of DBH is not unambiguously established. Finally, the mechanisms by which the protein binds the membrane are still unknown.

In this work, we analyzed the human DBH in an attempt to resolve these matters. The expression pattern of a full-length human DBH cDNA was compared with that of different mutated or truncated cDNAs in two mammalian cell lines using the vaccinia virus expression system.


MATERIALS AND METHODS

Plasmid Constructions

The vaccinia virus recombination vector (pgpt-ATA-18) used in this study is derived from pATA-18 vector (24) , into which a bacterial guanine phosphorybosyl transferase (gpt) gene driven by the vaccinia early/intermediate I3 promoter has been inserted (25) . The expression of the gpt gene allows efficient dominant selection of recombinant viruses (26) . The various DBH cDNAs were introduced downstream of the mutated vaccinia 11K late promoter at appropriate sites in the polylinker.

The pgpt-DBH-t construct (t for truncated) contains a human DBH cDNA (8), starting at nucleotide +4 of the coding sequence. The full-length cDNA construct (pgpt-DBH-f) was generated by adding the first ATG and the Kozak consensus (27) to pgpt-DBH-t plasmid. This was done by replacing the first 54 nucleotides by a double-stranded 59-mer oligonucleotide, thereby forming the complete sequence of the human DBH cDNA.

Mutations in the NH-terminal coding region of pgpt-DBH-f (full-length cDNA) were introduced by polymerase chain reaction (PCR) as described by Higuchi (28) . Two primary PCR reactions were performed with each of the complementary oligonucleotides containing a mutation and a 5`- or 3`-flanking oligonucleotide. The resulting PCR products were then combined and reamplified with the outside primers. The final secondary PCR products were subcloned into pgpt-DBH-f plasmid. Two mutants were generated: pgpt-DBH-m1, in which the second methionine codon is changed to a valine codon, and pgpt-DBH-m2, in which both the second and the third methionine codons are changed to valine codons. The mutagenic and the flanking primers were, respectively, a 20-mer (m1, 5`-CCCAGCGTGCGGGAGGCAGC-3`), a 33-mer (m2, 5`-CCCAGCGTGCGGGAGGCAGCCTTCGTGTACAGC-3`), a 22-mer (p-ATA-18, 5`-GTACGCTAGTCACAATCACCAC-3`), and a 20-mer (O2, 5`-ATAGGCAGTGTCCCCATCGG-3`). All constructions were confirmed by sequencing.

Generation of Recombinant Viruses

Recombinant vaccinia viruses were obtained by following the protocol of Stunnenberg and Schmitt (25) . Briefly, a human 143 cell line (TK) infected with wild-type vaccinia virus at a multiplicity of 0.1 plaque-forming unit per cell was transfected with pgpt-DBH precipitated with calcium phosphate. DBH cDNA was inserted into the virus by homologous recombination in the thymidine kinase gene. Recombinant viruses expressing DBH were selected by three consecutive rounds of plaque purification in the presence of mycophenolic acid, xanthine, and hypoxanthine (gpt-selective medium).

Expression of Dopamine -Hydroxylase in Mammalian Cells

RK 13 and AtT 20 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and Dulbecco's modified Eagle's medium:F12 (1:1), respectively, supplemented with 10% fetal calf serum. For infection with recombinant vaccinia virus, cells were plated the day before infection at 3 10 cells/100-mm tissue culture dish. Virus was added at a multiplicity of infection of 0.5 plaque-forming unit per cell. The infected cells were harvested after incubation for 14 h at 37 °C.

Subcellular Fractionation of AtT 20 and RK 13 Cells

Infected cell pellets were resuspended in 150 µl of 10 mM phosphate buffer, pH 7.0, 1 mM phenylmethylsulfonyl fluoride, and lysed by repeated freeze/thaw steps. The cell suspensions were centrifuged for 10 min at 100,000 g, and the supernatants (soluble fraction) containing cytoplasmic and lumenal proteins were collected. The pellets were washed three times with the same buffer. They were then resuspended in 150 µl of 10 mM phosphate buffer, pH 7.0, containing 0.5% Triton X-100 and centrifuged for 10 min at 100,000 g. The resulting supernatants were designated the solubilized membraneous fractions.

The soluble and membraneous as well as the washing fractions were analyzed by Western blotting and enzyme activity assays.

Dopamine -hydroxylase activity was assayed in whole cell extracts and subcellular fractions as previously described by Vayer et al.(29) .

N-Glycanase Digestion

Samples to be assayed for DBH deglycosylation were adjusted to 0.5% SDS and 0.1 M 2-mercaptoethanol and boiled for 3 min. The samples were then diluted with 0.2 M sodium phosphate buffer, pH 8.0, and digested with 10 units/ml N-glycanase (Genzyme) at 37 °C for 20 h.

Western Blot Analysis

Proteins in whole cell extracts or the subcellular fractions were separated by 5-12% polyacrylamide gel electrophoresis in the presence of SDS and transferred to nitrocellulose. Blots were incubated with a 500-fold dilution of a rabbit antiserum to human dopamine -hydroxylase (30) . Bound antibody was detected either with alkaline phosphatase-conjugated anti-rabbit IgG antibody or with I-labeled protein A. The autoradiograms were quantified by densitometry (Biocom). The soluble and membraneous DBH used as positive controls were prepared from human pheochromocytoma, as described by Roisin et al. (31).


RESULTS

Expression of Recombinant DBH Using Vaccinia Virus

The structural and functional properties of human DBH were analyzed using the vaccinia virus expression system. This viral vector is an efficient system for the expression of foreign genes in a wide range of host cells. Two different mammalian cell lines (AtT 20 and RK 13) were used. The mouse pituitary AtT 20 cells were chosen because they contain secretory vesicles, as do the chromaffin cells that naturally synthesize DBH. A comparative study was done with the rabbit kidney RK 13 cells, which are devoid of such vesicles.

The human DBH cDNA first reported by Lamouroux et al.(8) lacks the first initiation ATG codon. Consequently, the complete cDNA sequence was generated, and the full-length cDNA (DBH-f) as well as the truncated cDNA (DBH-t) were each inserted into a vaccinia virus by homologous recombination. In the case of the DBH-t construct, translation should start at the second ATG, which is surrounded by an appropriate Kozak consensus (27) . The resulting protein is expected to be produced with a signal sequence of 25 amino acids, whereas the native signal sequence is 39 amino acids long.

Whole cell extracts of AtT 20 and RK 13 cells infected with recombinant virus particles were analyzed by immunoblotting and assayed for DBH activity. The protein produced by the truncated cDNA migrated as a single homogeneous band with an apparent molecular mass of 73 kDa (Fig. 1B, lane3), while the full-length cDNA generated two distinct subunits of 73 and 77 kDa (lane4). Both recombinant proteins were slightly shifted downward as compared with the human pheochromocytoma membrane and soluble DBH positive controls (lanes2 and 5). This migration heterogeneity was due to species-specific differences of glycosylation (demonstrated below; see Fig. 4). These expression experiments confirm that the 73- and 77-kDa subunits are encoded by the same cDNA.


Figure 1: Expression of human DBH cDNAs by recombinant vaccinia virus. A, the 5`-nucleotide sequence of human DBH cDNA (8, 9) is shown. Arrows indicate the 5`-extremities of the full-length (DBH-f) and the truncated (DBH-t) cDNA, respectively. The first and the second ATG codons are shown in boldfaceletters. The corresponding predicted amino acid sequences are represented below. B, AtT 20 cells were infected with the different vaccinia virus constructs, incubated for 14 h, and harvested. Whole cell extracts were separated by 5-12% polyacrylamide gel electrophoresis in the presence of SDS, blotted, and reacted with antiserum to human DBH. Lane1, wild-type vaccinia virus-infected cell extracts; lane2, human pheochromocytoma membrane extracts; lane3, DBH-t recombinant virus-infected cell extracts; lane4, DBH-f recombinant virus-infected cell extracts; lane5, human pheochromocytoma soluble extracts.




Figure 4: Analysis of N-glycanase treatment of DBH by immunoblotting. Soluble and membrane human pheochromocytoma extracts and recombinant DBH-t- and DBH-f-infected AtT 20 cell extracts were incubated with N-glycanase at 37 °C for 20 h. Lanes1 and 2, untreated extracts of membrane pheochromocytoma (0.4 and 2 µg); lane3, untreated DBH-f-infected cell extracts; lane4, N-glycanase-treated membrane pheochromocytoma extracts (2 µg); lane5, N-glycanase-treated DBH-f-infected cell extracts; lane6, N-glycanase-treated DBH-t-infected cells extracts; lane7, N-glycanase-treated soluble pheochromocytoma extracts; lane8, untreated DBH-t-infected cell extracts; lane9, untreated soluble pheochromocytoma extracts.



The DBH-t and DBH-f extracts from both cell lines were enzymatically active (Fig. 3A). The activity of the enzyme produced by the DBH-f construct was three or four times lower than that obtained with DBH-t, although it was 1.5-fold more immunoreactive as measured by densitometry (Fig. 3B). These findings were consistently observed for at least three independent clones for each construct. The reason for this difference in specific activity is unclear, but might be due to different folding of the proteins.


Figure 3: Analysis of recombinant DBH produced in RK 13 and AtT 20 cells. RK 13 (blackbars) and AtT 20 (hatchedbars) cells were infected with DBH-t, DBH-f, DBH-m1, and DBH-m2 vaccinia constructs. Whole cell extracts were assayed for DBH enzymatic activity (histogramA) and subjected to Western blot analysis (histogramB). Determinations were done in duplicate. DBH immunoreactivity was quantified using a Biocom densitometer. One typical experiment is shown.



Investigation of an Alternative Translation Initiation Codon

The 5`-end of the coding region for the human DBH gene contains three in-phase ATG codons (9) . The two subunits may therefore be generated by the initiation of translation at different ATGs. Indeed, the two upstream codons have a favorable context for the initiation of translation (27) , whereas the more downstream ATG has a weak Kozak consensus but is nevertheless conserved in rat and bovine DBH (Fig. 7). We used site-directed mutagenesis to change the second and third methionine codons to valine residues. Western blots of the proteins extracted from cells expressing the mutated clones DBH-m1 and DBH-m2 gave two immunoreactive bands (Fig. 2, lanes2 and 3) with electrophoretic mobilities indistinguishable from those produced by the DBH-f construct (lane5). The valine substitutions affected neither the enzymatic activity of DBH nor the intensity of the doublet band (Fig. 3, A and B). Thus, the first ATG appears to be the only physiological initiator codon used for the translation of the two polypeptides.


Figure 7: The NH-terminal regions of bovine, rat and human DBH. Sequences are aligned to give the maximum homologies. Amino acid sequences are taken from the following references: bovine DBH (10-14), rat DBH (15), and human DBH (8, 9). Black and shadedboxes represent identical and similar residues, respectively. The positively charged (n) and the hydrophobic (h) regions (35, 36) are underlined. Arrows indicate the putative signal peptide cleavage sites of bovine DBH (10, 17, 18, 37).




Figure 2: Western blot analysis of mutated recombinant DBH. In recombinant DBH-m1, the second Met codon has been changed to a Val codon, and in recombinant DBH-m2 the second and the third Met have been changed to Val codons (underlined, boldfaceletters). AtT 20 cells were infected with DBH-t (lane1), DBH-m1 (lane2), DBH-m2 (lane3), and DBH-f (lane5). Human pheochromocytoma membrane extracts were used as control (lane4).



Deglycosylation of DBH

DBH is a glycoprotein bearing asparagine-linked oligosaccharides consisting of both high mannose and biantennary complex oligosaccharides (32, 33) . To determine whether the two bands of human DBH were due to a single polypeptide with two different glycosylation states, as has been proposed for bovine DBH (21) , soluble and membrane DBH controls and recombinant DBH-t and DBH-f were treated with N-glycanase. In all cases, the apparent molecular weight of each protein decreased, but the migration pattern was maintained for each treated sample (Fig. 4, lanes4-7). This clearly indicates that the difference between the two subunits is not due to the sugar moiety.

After deglycosylation, DBH expressed by the full-length cDNA (lane5) is indistinguishable from that of the endogeneous DBH (lane4), indicating that the recombinant protein is correctly matured.

Subcellular Localization of Recombinant DBH

We investigated the subcellular localization of both DBH-t and DBH-f in AtT 20 and RK 13 cells by fractionation. Both 77- and 73-kDa subunits of DBH-f were found by Western blot (Fig. 5) in the membrane fraction (lane5). In contrast, the soluble fraction contained only the 73-kDa subunit (lane4). DBH-t gave a 73-kDa band in both the soluble and membrane fractions (lanes3 and 4). No immunoreactivity was detected in the washing fractions (data not shown).


Figure 5: Western blot analysis of AtT 20 subcellular fractions. AtT 20 cells were infected with recombinant DBH-t and DBH-f vaccinia constructs. The cell extracts were fractionated as described under ``Materials and Methods'' and analyzed by Western blot. Lane1, human pheochromocytoma soluble extracts; lane2, soluble fraction of DBH-t-infected cells; lane3, membrane fraction of DBH-t-infected cells; lane4, soluble fraction of DBH-f-infected cells; lane5, membrane fraction of DBH-f-infected cells; lane6, human pheochromocytoma membrane extracts.



The ratio between the membrane-bound and soluble DBH was obtained by enzymatic activity measurements as well as protein quantification by densitometry. The proportion of DBH in the membrane fraction (relative to total DBH) was determined for each construct in the AtT 20 and RK 13 cell lines (Fig. 6). Both the enzymatic activity (56 ± 15%) and protein content (62 ± 16%) of membrane-bound DBH-f were significantly higher than those of the membrane-bound DBH-t (38.5 ± 9% and 44 ± 10%, respectively) in the AtT 20 cell line. The results obtained with the fractionated RK 13 extracts were similar (the enzymatic activity of the DBH-f membrane fraction was 57.5 ± 13% of the total DBH, and the amount of protein was 64 ± 15%, whereas for the DBH-t membrane fraction the corresponding values were 37 ± 8% and 42 ± 7%, respectively).


Figure 6: Distribution of DBH in subcellular fractions. Soluble and membrane-bound fractions of RK 13 (black) and AtT 20 (hatched) cells infected with DBH-t and DBH-f vaccinia constructs were assayed for DBH enzymatic activity and immunoreactivity. HistogramA shows the enzymatic activity of the membrane-bound fraction (expressed as a percent of total DBH enzymatic activity). HistogramB shows the corresponding relative amounts of DBH in the membrane-bound fraction, quantified by densitometry of autoradiograms. Data are mean ± S.E. (bars) values of four separate experiments. Statistical comparison of mDBH-t ratio versus mDBH-f ratio was made using Student's t test; p < 0.01



The DBH-m1 and DBH-m2 constructs were similarly analyzed. The ratio of membrane-bound and soluble forms were not different to that of DBH-f (data not shown).


DISCUSSION

In this study, the biochemical characteristics of the human DBH protein were analyzed by expressing different recombinant DBH-vaccinia virus clones in two cell lines. Expression of the full-length human DBH cDNA (DBH-f) resulted in the production of an active protein composed of 77- and 73-kDa monomers, electrophoretically indistinguishable from the native protein (22, 30) . This demonstrates that, as in the case of bovine DBH (16) , the two monomers arise from a single mRNA. A number of mechanisms could account for the synthesis of two subunits: differential glycosylation, initiation of translation at different ATG, and/or optional cleavage of the signal peptide.

Glycosidase treatment of cell extracts containing recombinant DBH-f maintained the presence of the two bands with an increase in the electrophoretic mobilities. This indicates that the human DBH molecular weight heterogeneity is not due to differences in carbohydrate composition, as it has been reported for bovine DBH (21) .

The use of alternative ATG codons was examined by constructing two recombinant mutated clones, in which DBH-f codons Met 2 or Met 2 and 3 were changed to Val codons. The expression pattern of the mutants was indistinguishable from that of the wild type (apparent molecular weight of the two monomer products, specific activity, kinetics of expression, and proportion of membrane-bound and soluble forms of the enzyme). These data clearly indicate that the first ATG is the only effective initiation site and, more specifically, that the 73-kDa subunit does not result from an alternative initiation at the second or third ATG.

A recombinant DBH clone lacking the first ATG (DBH-t) was constructed. In this case, the second ATG serves as initiator codon, and the signal peptide is reduced from 39 to 25 residues. The expression of such a cDNA in cells led to the synthesis of a single 73-kDa subunit, comigrating with the soluble DBH control. The resolution of the acrylamide gels was sufficient to exclude the possibility that an intermediate form migrating between 73 and 77 kDa went undetected. Deglycosylation of DBH-t protein confirmed that there was only a single type of subunit produced.

All together, these results offer compelling evidence that the DBH doublet arises from optional cleavage of the signal peptide, where the 73-kDa form is the completely processed DBH form and the 77-kDa form retains the signal peptide.

Moreover, our results show that when the signal peptide is shortened, its cleavage is rendered compulsory. This points to the important part played by the amino-terminal 14 amino acids in the regulation of the cleavage of the signal peptide.

The existence of DBH subunits that have conserved the signal peptide has been previously well established in species other than human. Amino-terminal sequence analysis of the bovine DBH (10, 19) demonstrated that the signal peptide is not always cleaved. Likewise, Feng et al.(34) reported that the full-length rat DBH mRNA, translated in a cell-free system in the presence of pancreatic microsomal membranes, produces a protein that retains the amino-terminal signal sequence. Fig. 7compares the amino-terminal peptide sequences of human, bovine, and rat DBH. They are all composed of the three domains typical of a signal peptide (35, 36) : an NH-terminal region, which is positively charged (n-region), a central hydrophobic part (h-region), and finally, a more polar carboxyl-terminal domain (c-region) with potential signal peptidase cleavage sites. Two of these cleavage sites appear to be functional in bovine DBH (10, 17, 18, 37) . However, this signal peptide is atypical in that it also exhibits features of uncleaved signal peptides of class II proteins (38) . Both n- and h-regions are much longer than their counterparts in cleaved signal peptides. The n-region is 10-20 amino acids long, depending on the species, rather than 1-5 amino acids in length. Similarly, the hydrophobic h-region is 20 residues long compared with the 7-15 amino acids in the classical model. These uncommon characteristics could explain why the signal peptide is not always removed. The behavior of the DBH-t construct clearly indicates that a truncated signal peptide promotes an efficient cleavage. Thus, a feature allowing the cleavage to be optional may be localized in the n-region. The amino-terminal extremities of the signal peptide in the three species differ both in length and in amino acids composition. The only noticeable shared characteristic is the presence of a conserved PXPS motif. The presence of these -turn-promoting residues could modify the folding of this long signal peptide, rendering the cleavage site less accessible or susceptible to cleavage.

DBH exists both in soluble and membrane-bound forms. We used the DBH-t and DBH-f constructs to investigate the role of the signal peptide in anchoring the protein to the membrane. Fractionation of the extracts from cells expressing the complete DBH cDNA indicates that all of the 77-kDa subunit is associated with the membrane. It was not detected in the soluble fraction. This result is consistent with sequence analysis of bovine DBH showing that only the membraneous DBH contains the signal sequence (10, 19) . It is therefore very likely that the uncleaved signal peptide is involved in attaching the protein to the membrane. However, when the signal peptide is completely cleaved, as in the case of the DBH-t construct, approximately one-third of the DBH was still found associated with the membrane fraction. This indicates that a second anchoring mechanism must exist independent of the signal peptide. Similarly, Gibson et al.(23) showed that the in vivo expression of a bovine DBH devoid of its signal peptide generates both membrane-bound and soluble forms of the enzyme.

A comparison of DBH-f and DBH-t activities indicates that the Triton-extractible fraction was significantly increased when the signal peptide was not cleaved (DBH-f), with regard to the short construct (DBH-t). Therefore, the two modes of membrane attachment co-exist and seem to be additive. According to our enzymatic activity measurements, 20% of total DBH (i.e. 37% of the membrane-bound fraction) would be anchored in the membrane by an uncleaved signal peptide, whereas the second attachment mechanism would participate for about 38%. This latter estimation is consistent with the 40% value found by Gibson et al.(23) for bovine DBH expressed in Drosophila cells. On the other hand, amino-terminal sequence analysis of purified bovine membraneous DBH indicates that approximately 20-30% of membrane-associated subunits contain uncleaved signal sequence (10, 19) .

Several questions still remain about the nature of the second anchoring mechanism, about the relationship between the two modes of attachment, and finally about the regulation of the ratio of soluble to membraneous DBH forms.

There have been several studies aimed at elucidating the mechanism of DBH membrane attachment. The most obvious possibilities, covalently bound lipids (myristylation, palmitylation, glycosyl-phosphatidylinositol attachment), the presence of an hydrophobic domain other than the signal peptide, and a putative membrane anchor protein, have been ruled out (22, 39, 40, 41) . However, phosphatidylserine (PS) has been suggested to bind DBH to membranes (19, 42, 43). This binding is noncovalent, dependent on pH and on vesicle PS availability, and remains irreversible in the absence of detergent. This latter fact implies that the proportion of PS membrane-bound DBH should be directly dependent upon the concentration of PS in the membrane and more precisely in the inner leaflet of the chromaffin granule membrane. The percent of DBH anchored by a mechanism other than the signal sequence is approximately the same (around 40%), whether or not the host cells possess secretory vesicles (AtT 20 and RK 13 cells in this study; Schneider 2 Drosophila cells in Gibson et al.(23) ). This might reflect the cotranslational capture of DBH by every available membrane in these heavy expression systems, resulting in an overestimation of membrane-bound forms compared to the situation in vivo. The determination of the precise localization of the membrane-bound DBH in all of these expression systems would therefore be valuable.

If we presume that phosphatidylserine is responsible for the alternate and irreversible anchoring mechanism, a simple model explaining the necessity for as well as the relationships between the two modes of attachment can be proposed. The PS-bound DBH may constitute a stock of membraneous DBH, whereas the signal peptide-attached DBH would be a supply of the soluble form. The phosphatidylserine sites would be saturated with soluble DBH to constitute a reserve of membraneous active DBH. On the other hand, the amount of soluble DBH would be controlled by a sharp regulation of the cleavage of the signal peptide. This hypothesis implies the presence of a signal peptidase or a specific endopeptidase inside the vesicles. Additionally, the signal peptide might also be cleaved in the endoplasmic reticulum as argued by two lines of evidence. A precursor-product relationship has been established between the 77- and the 73-kDa subunit of rat DBH (44, 45) . The conversion seems to occur in the secretory pathway, prior to the exit from the trans-Golgi apparatus (46, 47) . Moreover, the 73-kDa DBH polypeptide is produced in mammalian cells that do not possess synaptic-like vesicles (RK 13 cells). The proportion of molecules cleaved at this step may be random, depending on the accessibility of the cleavage site. It should be noticed that sequence analysis has identified two amino termini indicating two cleavage sites, separated by 3 amino acids, in the purified soluble bovine DBH (17, 18) . This amino-terminal heterogeneity could reflect the different compartments where the signal peptide is cleaved.

Finally, the physiological importance of the presence of both soluble and membrane-bound DBH is still unclear. The soluble form is released by exocytosis into various body fluids (plasma, lymph, saliva, and cerebral spinal fluid), whereas the membraneous DBH is recycled back into the cell. A possibility for the functional significance of the existence of the three interacting forms of DBH (two membraneous and one soluble) would be to exert a precise regulation of the amount of circulating DBH by controlling at once its source and its resorption.


FOOTNOTES

*
This work was supported by grants from the CNRS, INSERM, the Ministère de l'Enseignement Supérieur et de la Recherche, and Rhône-Poulenc Rorer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-69-82-36-46; Fax: 33-1-69-82-35-80.

The abbreviations used are: DBH, dopamine -hydroxylase; gpt, xanthine guanine phosphorybosyl transferase; PS, phosphatidylserine; n-region, NH-terminal region of a signal peptide, which is positively charged; h-region, a central hydrophobic part of a signal peptide; c-region, a more polar carboxyl-terminal domain of a signal peptide; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank J.P. Henry for providing human anti-DBH antibody. We are grateful to H. Stunnenberg and J. Schmitt for the pgpt-ATA 18 vector and the wild-type vaccinia virus and for helpful advice for constructing the recombinant viruses. We also acknowledge P. Ravassard for assistance in comparing amino acid sequences and all our colleagues for critically reading the manuscript.


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