INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

There is increasing consensus that protein degradation can play a key role in the control of protein function, with parameters such as covalent modification (36), intracellular localization (50), and regulated cleavage (7, 31) all capable of influencing turnover rates. In many cases this degradation is mediated by a multicatalytic proteinase referred to as the proteasome. For the majority of proteasome substrates so far described, degradation is regulated by ubiquitin-protein ligases that recognize specific sequences in proteins targeted for degradation. Subsequent addition of multimeric ubiquitin units represents the first stage in a process that ultimately leads to degradation of the substrate (5, 24, 26). There are a few noted examples where degradation by the proteasome does not involve ubiquitination (22, 25), including that of ornithine decarboxylase (ODC), the first enzyme in the catalytic conversion of ornithine to polyamines (20). A negative feedback loop is generated where polyamines induce translation of the protein antizyme, which binds to sequences in the amino terminus of ODC. Ensuing conformational changes expose a hydrophilic PEST domain in the carboxy-terminal region that targets ODC for degradation directly by the proteasome (20, 28).

Numerous studies have identified factors that promote degradation of specific proteins by the proteasome; however, there are fewer reports of stimulants that inhibit degradation, either specifically or generally (24). In a rare example, it has been shown that internalized insulin can bind a proteasome activator called insulin-degrading enzyme and, in so doing, inhibit general protein metabolism (13, 19). However, there is no evidence to suggest that insulin specifically promotes the transcription or translation of factors that are capable of inhibiting the degradation of a small group of related proteins or that it could do so through activation of recognized signal transduction pathways.

Histamine is a biogenic amine that serves a number of important biological functions, including stimulation of acid secretion within the stomach (39, 45). It is generated through the action of the enzyme L-histidine decarboxylase (HDC; EC 4.1.1.22), which is initially translated as a 74-kDa protein but is subsequently cleaved to a number of smaller isoforms (8, 23). Recombinant experiments showed that a 54-kDa carboxy-terminally truncated HDC isoform had much greater activity than the primary translation product, leading to the general belief that such a regulated step in vivo would facilitate the production of an enzymatically active homodimer of 100 to 110 kDa (8, 42, 46, 47). However, other groups were unable to confirm that the 54-kDa isoform had a higher specific activity (48), and the presence of other isoforms has to some extent been ignored.

While the physiological relevance of multiple cleavage steps has not yet been fully deduced, studies on the rat protein sequence suggest the presence of a number of functional domains within the enzyme. This includes a putative intracellular targeting domain, located somewhere near the carboxy-terminal tail (44, 47, 48). Computer analysis has also identified two putative PEST domains at either end of the protein, which have been hypothesized to regulate degradation (15, 29, 43). While one or more of these regions could be cleaved off during posttranslational processing, specific cleavage sites have not yet been identified, and only carboxy-terminal cleavages have previously been considered. Recent studies have shown that the 74-kDa form of HDC can be degraded through the ubiquitin-proteasome pathway (43). While the contribution of this pathway to HDC activation has not yet been considered seriously, it is noteworthy that partial degradation of proteins by this pathway has, in some cases, been shown to regulate activation (30, 31).

Gastrin stimulation of enterochromaffin-like (ECL) cells in the gastric mucosa leads to activation of cholecystokinin B-gastrin receptor signaling shortly after feeding. This is followed by rapid release of histamine, which is then replenished through increased expression of HDC (3, 10, 12, 18, 32, 39). This upregulation of HDC occurs in part through increases in HDC mRNA abundance (3, 10, 11). However, recent studies additionally suggest some degree of posttranscriptional regulation, due both to the rapidity of the response and to the absence of inhibition by actinomycin D (3, 4, 21). These studies also showed that cycloheximide was able to block enzyme activation, suggesting that gastrin-dependent increases represent a primarily translational response (4, 9, 21). Indirect support for these proposals has come from studies which suggest that 5' untranslated region (5'UTR) sequences can promote HDC translation (23). Indirect support has also come from studies which showed that gastrin is able to stimulate translation of ODC mRNA through 5'UTR sequences and an eIF4E-dependent pathway (33).

In order to investigate the posttranscriptional regulation of HDC by gastrin, we subcloned the rat HDC cDNA into a eukaryotic expression vector and, after transfection into Cos-7 cells expressing the CCK-B/gastrin receptor, examined responses to gastrin stimulation. Our results suggest that transcription-independent increases in enzyme activity are unlikely to involve increases in HDC translation, mediated by the 5'UTR. Instead, we propose that gastrin stabilizes HDC isoforms that are generated through a novel combination of amino- as well as carboxy-terminal cleavage steps. We demonstrate that two PEST domains located within the enzyme mediate this stabilization and confirm that PEST domains from other rapidly turned-over proteins share this type of gastrin regulation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmid DNA constructs. Unless otherwise stated, all expression constructs were generated by PCR amplification using pfu DNA polymerase (Stratagene) on a thermocycler (Perkin Elmer 9700) and using the CMV-HDC18 vector (23) as the template. Amplification parameters for 30 cycles were as follows: 94°C for 30 s, 53°C for 45 s, and 72°C for 2 min per kb of PCR product. PCR products were initially blunt-end cloned into the pCRscript vector (Stratagene). Inserts were subsequently subcloned into appropriate expression constructs.

Cell culture. Cos-7 cells were maintained in complete medium, which consisted of Dulbecco's modified Eagle's medium (DMEM; BioWhittaker) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Life Technologies). Cells were cultured in a humidified incubator with 5% CO₂ at 37°C.

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Isolation of stomach tissue from rats. Male Sprague-Dawley rats weighing 200 to 250 g were fasted with free access to water; 48 h later, standard dietary nuts were added to cages containing test animals, and the rats were allowed to feed ad libitum. Control and test rats were sacrificed 3 h later by lethal injection (100 ng of ketamine-HCl per 100 g) and cervical dislocation. Whole stomachs were isolated and cleaned in phosphate-buffered saline (PBS), (136 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄). The tissue was homogenized in ice-cold 0.1 M sodium phosphate buffer (pH 7.4) containing 0.2 mM dithiothreitol. The tissue homogenate was centrifuged at 10,000 × g for 15 min, and the supernatant was transferred to a fresh tube. Protein content was estimated by the method of Bradford (Bio-Rad) for subsequent fractionation. Experiments were performed in accordance with local animal welfare regulations.

Assay of HDC activity. Cells were harvested in 2 ml of PBS, with a 500-µl aliquot removed for RNA analysis. The remaining cells were assayed for HDC activity using an adaptation of previously described methods (8). Briefly, cells were pelleted and resuspended in ice-cold 0.1 M sodium phosphate buffer (pH 7.4) containing 0.2 mM dithiothreitol and sonicated for 10 s. Whole-cell extracts were assayed for HDC activity by incubating 40-µl aliquots with 40 µl of 2× reaction buffer (2 µCi of L-[¹⁴C]histidine [Amersham], 0.5 mM L-histidine, 0.01 mM pyridoxal phosphate, 0.1 M sodium phosphate [pH 6.8]). Reactions were performed in an open microcentrifuge tube placed in a closed scintillation vial. ¹⁴CO₂ generated by the enzymatic reaction was trapped in 50 µl of 80% (vol/vol) Soluene 350 (Packard). The enzyme reaction was stopped by the addition of 50 µl of 3 M perchloric acid, and the reaction was incubated at room temperature for 30 min. Levels of radiolabeled CO₂ were determined by liquid scintillation counting. All samples were normalized to total protein content, and the protein concentration was determined by the method of Bradford.

Assay of luciferase activity. Cells were harvested in 2 ml of PBS, with a 500-µl aliquot removed for RNA analysis. The remaining cells were lysed in 1× cell lysis buffer and assayed for luciferase activity as advised by the manufacturer (Promega). All samples were normalized for protein content, and protein concentration was determined using a detergent-compatible protein estimation kit (Bio-Rad).

RNA analysis. Cells to be analyzed for RNA were pelleted and stored at 70°C until required. RNeasy kits (Qiagen, La Jolla, Calif.) were used to extract total RNA from in vitro-cultured cells. Total RNA was fractionated on 1% agarose denaturing formaldehyde gels, and the RNA was blotted to nylon membranes (Amersham Pharmacia Biotech) using established capillary blotting methods. DNA probes for Northern blot analysis were labeled with [-³²P]dCTP (3,000 mCi/mmol; NEN) using a random primer labeling kit as advised (Megaprime; Amersham). A full length (2.36 kb) HDC cDNA fragment generated by EcoRI and SalI digestion of the pEP-HDC2.4 vector was used as the template for an HDC-specific probe. For luciferase-specific hybridizations, a 500-bp fragment generated by HindIII and EcoRV digestion of the pGL75 vector was used as the template for labeling reactions. For glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a commercially available human G3PDH probe was used (Clontech). Hybridizations were performed at 65°C using Quickhyb solution (Stratagene) following the manufacturer's instructions, and membranes were washed to high stringency in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) at 65°C. After exposure to X-Omat LS autoradiographic film (Kodak), blots were stripped with 0.1% SDS at 95°C for 5 min before rehybridization with other probes. Quantitation was performed using appropriate computer software (NIH Image).

Generation of an anti-HDC antibody. A custom-prepared anti-HDC antibody was raised in a rabbit and affinity purified (Immunodynamics). Peptides corresponding to residues 30 to 44, 133 to 147, and 321 to 335 of the rat HDC protein sequence were used for immunizations. To confirm the specificity of the antibody, lysates from Cos-7 cells transfected with the pEP-empty vector were compared with lysates from cells transfected with either pEP-HDC1.5 or pEP-HDC2.0 as described in the text (see Fig. 6C). Comparisons were performed using both Western blotting and immunoprecipitation methods.

Western blotting analysis. Cell and tissue lysates were diluted in 2× sample buffer (130 mM Tris-HCl containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.1% bromophenol blue), boiled for 5 min, and electrophoresed on SDS-polyacrylamide (10%) gels. Fractionated proteins were transferred electrophoretically (60 mA) to a polyvinylidene difluoride membrane (NEN Dupont) in 24 mM Tris buffer containing 40 mM glycine and 20% methanol. The transfer was performed overnight at 4°C. Membranes were blocked in PBS containing 1% Tween 20 (PBS-T) and 5% nonfat milk overnight at 4°C. The membranes were washed briefly with room temperature PBS-T before the addition of primary antibodies. Anti-HDC antibody was added at a dilution of 1:20,000 in PBS-T containing 5% nonfat milk, while the anti-blue fluorescent protein (anti-BFP) antibody (Clontech Living Colors) was added at a dilution of 1:1,000 in PBS-T containing 1% bovine serum albumin. After 2 h, the membranes being probed for HDC were washed three times for 20 min at 39°C in PBS-T, while membranes being probed for BFP were washed at room temperature. After washing, the blots were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Amersham) at a dilution of 1:1,000 in PBS-T containing 1.0% bovine serum albumin. The membrane was washed three times in PBS-T at room temperature, and immunoreactive proteins were detected using the Renaissance kit (NEN). Quantitation was performed using appropriate computer software (NIH Image).

Immunoprecipitation of HDC isoforms and GFP-PEST chimeras. Cos-7 cells were seeded at a density of 10⁶ cells per 100-mm dish and cotransfected with 5 µg of pEP-GR and 20 µg of pEP-HDC2.4, pGF-PEST-ODC, or pGF-PEST-DDC as described above. Twenty-four hours after removal of the transfection mix, the cells were washed twice with PBS and incubated in cysteine- and methionine-free DMEM (Cys/Met medium; BioWhittaker) supplemented with 10% dialyzed fetal bovine serum (Gibco), 2 mM L-glutamine, and 1% penicillin-streptomycin solution (Gibco). After 1 h the medium was replaced with Cys/Met medium supplemented with actinomycin D (20 µg/ml, final concentration) and 200 µCi of Easytag Express ³⁵S-labeled methionine-cysteine mix (1,175 mCi of [³⁵S]methionine per mmol; New England Nuclear). After a 4-h pulse, cells were washed twice with PBS, and complete medium supplemented with actinomycin D (20 µg/ml, final concentration) was added. For test cells, gastrin (10⁷ M) or lactacystin (10⁵ M) was added. Cells were harvested at appropriate time points in 1 ml of radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 2 mM EDTA, 0.1% SDS, 0.25% deoxycholate, 1% Triton X-100) supplemented with protease inhibitors (Boehringer Mannheim). After a minimum incubation of 1 h on ice, cell lysates were centrifuged at 12,000 × g for 10 min at 4°C, and a 950-µl aliquot of the supernatant was added to 30 µl of protein A-Sepharose CL-4B (Pharmacia; 30 mg/ml in PBS). Samples were incubated overnight at 4°C with constant agitation. Precleared samples were centrifuged for 1 min at 12,000 × g, and a 900-µl aliquot of supernatant was transferred to a fresh tube containing 2 µl of affinity-purified anti-HDC antibody or 4 µl of polyclonal anti-GFP antibody (Molecular Probes). After a 1-h incubation at 4°C, 30 µl of protein A-Sepharose CL-4B was added, and samples were incubated for a further hour. Samples were centrifuged at 12,000 × g for 1 min, and the resulting precipitate was washed four times with 1 ml of RIPA buffer. The pellet was diluted in an equal volume of 2× sample buffer, and radiolabeled HDC isoforms were fractionated by electrophoresis on SDS-polyacrylamide (10%) gels. Gels were dried under vacuum and exposed to Biomax MR film using appropriate LS intensifying screens (Kodak). Quantitation was performed using appropriate computer software (NIH Image).

Intracellular localization of GFP-HDC chimeras. Cos-7 cells were seeded at a low density on poly-D-lysine-coated glass microscope wells. The next day, the cells were transfected for 2 h with 20 µg of the pGF constructs (pGF-empty, pGF-ER1, pGF-ER2, pGF-antiER2, and pHDC2.0-GF) and Superfect reagent, as advised by the manufacturer (Qiagen). After 24 h, ER-Tracker Blue-White DPX was added to the cells at a concentration of 100 nM in complete medium as advised by the manufacturer (Molecular Probes). The cells were incubated for a further 30 min at 37°C before being washed twice with PBS and visualized at ×100 and ×60 magnification by fluorescent microscopy.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

HDC activity is stimulated by gastrin in a transcription-independent manner. In order to examine the posttranscriptional regulation of HDC, we have generated an HDC expression construct (pEP-HDC2.4) that codes for the rat HDC protein. The construct contains the complete HDC cDNA sequence cloned downstream from the CMV promoter and upstream from the simian virus 40 polyadenylation signal. The inserted fragment includes 75 bp of 5'UTR, 317 bp of 3'UTR, and 1,968 bp of coding sequence (total, 2.36 kb).

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View larger version (15K): [in this window] [in a new window]   FIG. 1.   Time course showing the effect of gastrin stimulation on HDC enzyme activity and HDC mRNA levels. Cos-7 cells transfected with pEP-HDC2.4 and pEP-GR were treated for 2 h with actinomycin D and stimulated in a reverse time course with gastrin (107 M) for the time periods shown. (A) Effect of stimulation on HDC enzyme activity. Data show the combined results from four independent experiments ± SEM. (B) Northern blot showing the expression of HDC mRNA after gastrin stimulation. -ve, expression in cells transfected with the pEP-empty vector and stimulated with gastrin for 5 h. G3PDH shows relative loading. The 18S and 28S arrows indicate the migration of rRNA subunits. The graph shows HDC expression corrected for loading. Similar Northern blotting results were obtained in three other experiments.

Gastrin stimulation of HDC involves activation of PKC and MEK1 pathways. We next wanted to identify which intracellular signalling pathways are involved in this gastrin effect. Cells transfected with pEP-HDC2.4 and pEP-GR were pretreated for 2 h with actinomycin D and stimulated with forskolin, PMA, and thapsigargin in order to activate protein kinase A (PKA), protein kinase C (PKC), and intracellular calcium release, respectively. The results from these experiments, which are shown in Fig. 2A, indicated that gastrin-induced increases in HDC activity were most closely mimicked by stimulation with 10⁸ M PMA, with levels increased from 1.04 ± 0.18 nmol/mg/h to 3.5 ± 0.65 nmol/mg/h. Stimulation with either thapsigargin or forskolin had no significant effect on HDC activity. Northern blotting confirmed that HDC mRNA levels were unaltered by treatment with these compounds (Fig. 2B).

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Effect of signaling pathway activators and inhibitors on HDC enzyme activity. (A and B) Cos-7 cells transfected with pEP-HDC2.4 and pEP-GR were treated for 2 h with actinomycin D and stimulated for 4 h with 107 M gastrin, 104 M forskolin (Forsk.), 108 M PMA, or 106 M thapsigargin (TG). -ve, cells transfected with the pEP-empty vector. Control, unstimulated cells transfected with pEP-HDC2.4. (A) Effect of stimulation on HDC enzyme activity. Data are the means of three independent experiments ± SEM. (B) Northern blot showing the expression of HDC mRNA after stimulation with the signaling pathway activators shown in A. G3PDH shows relative loading. The graph shows HDC expression corrected for loading. Similar Northern blotting results were obtained in three other experiments. (C) Cos-7 cells transfected with pEP-HDC2.4 and pEP-GR were treated for 2 h with actinonmycin D and specific inhibitors; PKC inhibitor staurosporine (Stauro., 50 µM), MEK1 inhibitor PD98059 (20 µM), or the translation inhibitor cycloheximide (CHX, 20 µg/ml). After 2 h of treatment, the cells were stimulated for 4 h with gastrin (107 M). Data, which show the fold increase in HDC enzyme activity after gastrin stimulation, are the combined results from three independent experiments ± SEM.

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Gastrin stimulation leads to increased levels of HDC isoforms. A total of five HDC isoforms have so far been identified in rat tissue extracts. This includes the full-length 74-kDa primary translation isoform, a 63-kDa isoform, two isoforms having molecular masses close to 54 kDa, and a smaller isoform of about 36 kDa (8). We wished to investigate the molecular basis for transcription-independent increases in HDC activity and consequently developed an affinity-purified polyclonal antibody to study HDC expression at the protein level. The antibody was raised against three peptide regions in the amino terminus of rat HDC, and its specificity was confirmed by immunoprecipitations and Western blots (see Materials and Methods section).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   HDC isoform expression in transfected Cos-7 cells and in rat stomach cell lysates. (A) Cos-7 cells transfected with pEP-HDC2.4 and pEP-GR were treated for 2 h with actinomycin D and then stimulated with 107 M gastrin for 4 h. Western blots were probed for HDC isoform expression using an anti-HDC antibody raised against antigens in the amino terminus of HDC. Immunoreactive HDC isoform expression was detected after 10 and 30 min of exposure of blots to autoradiographic film. The results shown are representative of four independent experiments. Asterisks indicate secondary bands as described in the text. (B) Rats that had been fasted for 2 days were given free access to food for 3 h. HDC isoform expression in the stomach cell lysates of fasted and refed animals was analyzed by Western blotting. The results shown are representative of three independent experiments performed on paired fasted and refed rats.

5'UTR of HDC mRNA does not mediate a gastrin-stimulated increase in mRNA translation. Gastrin stimulation of HDC activity in Cos-7 cells is sensitive to cycloheximide treatment (Fig. 2C) and leads to an increase in the levels of the primary 74-kDa HDC translation product (Fig. 3A). Therefore it was hypothesized that gastrin could act to increase the translation of HDC mRNA. Previous studies have shown that gastrin can act via 5'UTR sequences and in a cycloheximide-sensitive manner to increase the translation of ODC mRNA, strengthening the rationale for this hypothesis (33).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Effect of gastrin stimulation on 5'UTR-mediated translation. (A) Diagrammatic representation of 5'UTR-luciferase (Luc.) constructs containing either 1 bp (pGL-01) or 75 bp (pGL-75) of HDC 5'UTR. (B) Cos-7 cells were transfected with gastrin receptor (pEP-GR) and 5'UTR-luciferase (pGL-01 and pGL-75) constructs. Cells were treated for 2 h with actinomycin D and cultured in the presence or absence (control) of 107 M gastrin for 4 h. The graph shows the effect of gastrin stimulation on absolute luciferase activities in a typical experiment (mean ± range for two different transfections), and data are representative of six independent experiments. RLU, relative light units.

Gastrin stimulation stabilizes HDC isoforms. Having demonstrated that gastrin stimulation of the 5'UTR is unlikely to increase HDC translation, we wanted to investigate whether increased isoform expression arises as a result of increased protein stability. In order to test this, we pulse-labeled transfected Cos-7 cells for 4 h, and the pattern of HDC isoform degradation was chased in the presence and absence of gastrin. Protein lysates from pulse-labeled cells were subjected to immunoprecipitation using our anti-HDC antibody, which confirmed the expression of multiple HDC isoforms (Fig. 5A). However, some of the isoforms were more weakly detected than by Western blotting, presumably reflecting differences in the affinity of the antibody for the native isoforms in solution.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Pulse-chase showing the effect of gastrin stimulation on HDC isoform stability. (A) Cos-7 cells transfected with pEP-HDC2.4 and pEP-GR were pulse labeled with 35S-labeled methionine and cysteine in the presence of actinomycin D. After a 4-h pulse, cells were cultured in control medium in the absence (control) and presence of 107 M gastrin for the time intervals shown. HDC isoforms were immunoprecipitated and electrophoretically fractionated. (B) Graphs showing the effect of gastrin stimulation on the degradation of the 48-kDa and 63-kDa HDC isoforms. Each point represents the mean ± SEM for three independent experiments. Values for isoform half-lives (t1/2) represents the mean ± SEM of values determined for each of the three individual experiments. Statistically significant differences relative to the corresponding control values are indicated by a superscript a (P < 0.05, Student's t test).

Distinct domains in the primary protein sequence regulate HDC expression levels. Gastrin stimulation increased the half-lives of both the 48- and 63-kDa isoforms (Fig. 5); however, in view of the fact that these increases occurred in the setting of different basal rates of protein turnover, it appeared likely that expression of the different HDC isoforms can be differentially regulated. There was some evidence for this in gastrin-stimulated Cos-7 cells, where the increase in expression of the 36-kDa isoform appeared greater than increases observed in expression of some of the other isoforms (Fig. 3A). Refeeding of fasted rats also appeared to preferentially increase the expression of the smaller HDC isoforms in rat stomach cell lysates. For example, we noted a 3.5- ± 1.0-fold increase in the 48-kDa isoform, compared to a 1.7- ± 0.5-fold increase in the 74-kDa isoform (mean ± SEM, n = 3, Fig. 3B). This led us to believe that there are domains present in some but not all isoforms which are capable of differentially regulating enzyme stability and protein expression levels.

View larger version (29K): [in this window] [in a new window]   FIG. 6.   Effect of carboxy-terminal truncations on HDC expression. (A) Diagrammatic representation of the carboxy-truncated constructs used in panels B, C, and D. Predicted sizes of the primary translation products are shown on the right-hand side. Amino acid numbers in the primary rat sequence are shown underneath. (B, C, and D) Expression constructs pEP-HDC2.0, pEP-HDC1.7, pEP-HDC1.6, and pEP-HDC1.5 were transiently transfected into Cos-7 cells. After 48 h, the cells were harvested for (B) HDC enzyme activity analysis, (C) HDC protein expression analysis by Western blotting, and (D) HDC mRNA expression by Northern blotting. G3PDH shows relative loading. -ve, cells transfected with the pEP-empty vector. The enzyme activities (B) and Western blot (C) are derived from the same experiment and are representative of six independent experiments. Enzyme activities show mean ± range for two readings from the same sample. The Northern blot is representative of four independent experiments.

Amino acids 568 to 656 of the rat HDC protein encode an endoplasmic reticulum signaling sequence. Cell fractionation experiments generally suggest that the 74-kDa and 53- or 54-kDa rat HDC isoforms are differentially localized within the cell, although contradictory results have been reported (44, 47). Because these earlier studies assumed that the 54-kDa isoform is generated as a consequence of carboxy-terminal cleavage only, it has been proposed that amino acids 485 to 656 of the rat protein sequence are involved in intracellular targeting (8, 44).

View larger version (41K): [in this window] [in a new window]   FIG. 7.   Localization of fluorescent chimeras. Cos-7 cells were transfected with the expression constructs pGF-empty (A), pHDC2.0-GF (B), pGF-ER2 (C), pGF-antiER2 (D), and pGF-ER1 (E), as diagrammatically represented with numbered amino acids from the HDC protein sequence. Panels A and B and the left-hand panels of C, D, and E show patterns of intracellular localization of GFP chimeras 24 h after transfection. The right-hand panels of C, D, and E show endoplasmic reticulum localization of the ER-Tracker probe in the cells shown in the corresponding left-hand panels. The magnifications and exposure times are shown underneath the photographs.

PEST domains of HDC regulate protein expression levels in a gastrin-responsive manner. Our results in Fig. 6 showed that the HDC sequence between amino acids 517 and 568 also regulates protein expression levels. This region corresponds in part to a PEST domain previously identified by computer analysis (15) and is one of two such regions identified in the primary rat HDC protein sequence (Fig. 6A). To study the role of this PEST domain, PEST2, in regulating HDC enzyme expression and to test whether this region might mediate gastrin regulation of isoform expression, we fused the PEST2 peptide in frame to the carboxy terminus of the BFP (Fig. 8A, BFP-PEST2). This pBF-PEST2 construct was transiently transfected into Cos-7 cells, and after actinomycin D treatment, the cells were stimulated with 10⁷ M gastrin. Western blotting with an antibody raised against the BFP was used to examine protein expression levels.

View larger version (13K): [in this window] [in a new window]   FIG. 8.   Effect of gastrin stimulation and proteasome inhibition on BFP-PEST expression levels. (A) Diagrammatic representation of BFP-PEST2 and BFP-PEST1, with numbered amino acids from the primary rat HDC protein sequences shown underneath. (B, C, D, and E) Cos-7 cells were cotransfected with pEP-GR and either pBF (B and C), pBF-PEST2 (B and D), or pBF-PEST1 (C and E). Cells were treated for 2 h with actinomycin D before incubation for 4 h with 107 M gastrin (B and C) or for 6 h with 105 M lactacystin (D and E), a proteasome inhibitor. Control cells were left untreated (B, C, D, and E). Western blotting using an anti-BFP antibody was used to examine the patterns of chimeric protein expression. The results shown are representative of not less than three independent experiments, as detailed in the text.

Gastrin stimulation stabilizes chimeric proteins that contain the PEST domains from ODC and DDC. The experiments shown in Fig. 8 demonstrated that gastrin disproportionally increases the expression of sequentially unrelated HDC-PEST domain chimeras, with the most rapidly turned-over BFP-PEST2 protein also being the most stimulated by gastrin. Consequently it was proposed that gastrin might regulate the stability of other rapidly turned-over PEST domain-containing proteins. To test this, we cloned the PEST domains of two other decarboxylase enzymes, ODC and DDC, and fused them in frame to the carboxy terminus of GFP (pGF-PEST/ODC and pGF-PEST/DDC) (Fig. 9A). The effect of gastrin stimulation on GFP-PEST fusion protein stability was examined in pulse-chase experiments. These experiments confirmed that gastrin was able to regulate the stability of other PEST domain-containing proteins, although to differing degrees (Fig. 9B). For the GFP-PEST/ODC protein, this gastrin regulation led to a 2.3- ± 0.5-fold increase in expression after a 6-h chase (mean ± SEM, n = 3; top panel, lane 2). Similar increases in GFP-PEST/ODC expression were observed when the proteasome was inhibited by lactacystin (2.6- ± 0.2-fold, mean ± SEM, n = 3; top panel, lane 3). Gastrin stabilization of the GFP-PEST/DDC fusion protein was less marked after the 6-h gastrin chase (1.4- ± 0.1-fold increase in expression; mean ± SEM, n = 3; middle panel, lane 2), and lactacystin treatment had no detectable effect on expression levels (1.1- ± 0.3-fold, mean ± SEM, n = 3; middle panel, lane 3).

View larger version (17K): [in this window] [in a new window]   FIG. 9.   Effect of gastrin stimulation and proteasome inhibition on the stability of GFP chimeras containing the PEST domains of ODC and DDC. (A) Diagrammatic representation of GFP-PEST/ODC and GFP-PEST/DDC, with numbered amino acids from the primary rat ODC and rat DDC protein sequences shown underneath. (B) Cos-7 cells transfected with pEP-GR and either pGF-PEST/ODC, pGF-PEST/DDC, or pGF-empty vector were pulse labeled with 35S-labeled methionine and cysteine in the presence of actinomycin D. After a 4-h pulse, cells were chased in control medium supplemented with 107 M gastrin or 105 M lactacystin for 6 h. Control cells were left untreated. GFP chimeras were immunoprecipitated and electrophoretically fractionated on a single gel. The results shown are derived from a common autoradiograph (i.e., common exposure time) from a representative experiment (n = 3).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Here we establish that the peptide hormone gastrin acts through G protein-stimulated pathways to regulate the stability of a group of rapidly turned-over PEST domain-containing proteins. The PEST domains used to prove this are sequentially unrelated other than that they define hydrophilic regions in their native proteins. This argues against a direct effect at the level of substrate recognition but instead suggests that gastrin inhibits some common component of the general protein degradation machinery. We further demonstrate that the PEST domain proteins most susceptible to this gastrin stabilization are also the proteins most affected by proteasome inhibition. This is again supportive of a gastrin effect on protein degradation, with rapidly turned-over PEST-containing proteins the most affected. The HDC enzyme, which contains PEST domains at both the amino- and carboxy-terminal ends, provides a unique model with which to study this type of hormonal regulation. Our results indicated that gastrin stimulation leads to increased HDC enzyme activity and that this transcription-independent increase requires the activation of PKC and MEK1 pathways. Although regulation is dependent upon novel protein synthesis, our experiments showed that it is unlikely to relate to increased translation of HDC. Instead, our results point to a dependence on the translation of one or more protein factors capable of inhibiting the degradation of HDC and other PEST domain-containing proteins. To our knowledge, this is the first report of a hormone regulating the degradation of multiple PEST domain-containing proteins and thus delineates a novel mechanism of hormone regulation of protein function.

In this study we characterized two discrete and transferable HDC PEST domains which promote degradation of heterologous proteins and regulate stabilization by gastrin. However, our studies identified additional features of HDC regulation that are likely to determine whether these domains are buried within the primary structure or exposed flush against the end of the enzyme. In particular, we identified a key role for the carboxy-terminal ER2 domain in intracellular localization and patterns of protein processing. First, we found that carboxy-terminal truncation to sequentially remove the ER2 and PEST2 domains successively increased expression of HDC protein, with corresponding increases in enzyme activity. While PEST2 regulation of protein expression might have been expected based on studies with other rapidly degraded proteins (35), it was not anticipated that removing the C-terminal 87 amino acids would, by itself, also lead to increases in protein expression levels. Fluorescent protein chimeras containing this 87-amino-acid region colocalize with endoplasmic reticulum-specific probes, suggesting that ER2-mediated targeting may be associated with degradation of the 74-kDa primary HDC translation product. This degradation is known to occur via the ubiquitin-proteasome pathway (43), in a process that is likely to involve ubiquitin-conjugating enzymes. Many of these conjugation enzymes have been localized to the endoplasmic reticulum membrane (1, 40), and thus an endoplasmic reticulum-localizing sequence could hypothetically target the 74-kDa isoform for conjugation and degradation. It is interesting that NF-B activation involves partial degradation of the p105 isoform by the ubiquitin-proteasome pathway (7, 30, 31), raising the possibility of partial degradation and subsequent stabilization and activation of HDC by this mechanism.

The carboxy-terminal ER2 targeting domain also appears to influence protein processing, and it was noted that truncation of HDC to exclude this region led to substantially altered cleavage patterns (see Fig. 6C for processing of pEP-HDC1.5, -1.6, and -1.7 vector products). In the first instance, it was anticipated that carboxy-terminal truncations to generate stable HDC proteins would also lead to increased levels of the smaller processed HDC isoforms. We found no evidence for this, suggesting that generation of processed isoforms either directly or indirectly involves the ER2 region.

Our experiments with truncated HDC proteins also suggested that the carboxy-terminal ER2 domain somehow inhibits an amino-terminal cleavage, which is a novel form of processing not previously considered for HDC. Specifically, it was noted that expression of the full-length protein led to the detection on Western blots of a protein of the predicted 74-kDa size. In contrast, when we expressed truncated proteins that lacked the carboxy-terminal ER2 domain, the major protein band detected was about 4 to 5 kDa shorter than anticipated. Northern blots confirmed HDC transcripts of the correct sizes, and Western blots confirmed that the predicted pattern of carboxy-terminal truncations had been maintained. This suggested that discrepancies between predicted and actual sizes arise as a consequence of amino-terminal cleavage, which occurs shortly after translation. These results therefore provided preliminary evidence for an amino-terminal processing step which is promoted when the ER2 domain is either engineered to be absent (as was done here) or removed by cleavage (as is likely to occur in vivo).

Experiments with the PEST1 fluorescent chimera provided further evidence for this type of regulation. In addition to detecting the primary ~45-kDa primary translation product for BFP-PEST1, low levels of a smaller specific band of ~35 kDa were also detected. This band is slightly larger than the BFP protein, indicating the presence of additional HDC protein sequence, again consistent with an amino-terminal processing step.

Many proteins contain short amino-terminal sequences that are cleaved in response to specific stimuli (37) or following translocation across the endoplasmic reticulum (6, 41), and we confirm here (Fig. 7E) that amino acids 1 through 40 (hereafter referred to as ER1) are at least capable of targeting a chimeric fluorescent protein to the endoplasmic reticulum. The presence of the carboxy-terminal ER2 domain therefore appears to inhibit an amino-terminal cleavage that removes the ER1 domain. We conclude that more than one processing pathway is available for the primary translation product depending on whether or not the carboxy-terminal ER2 is present. It is conceivable that tissue-specific factors will regulate whether amino- or carboxy-terminal processing patterns dominate, which might explain why some of the isoforms identified in transfected Cos-7 cells were not as clearly detected in rat stomach cell lysates. It might also explain why only 74-kDa and 54-kDa HDC isoforms have been reported in rat basophilic cells (43).

Interestingly, an amino-terminal cleavage such as that described here would place the amino-terminal PEST domain flush against the end of the enzyme, potentially altering its susceptibility to degradation or, for that matter, regulation by gastrin. We have considered cleavage steps that would initially expose and then remove the PEST domains at both the amino- and carboxy-terminal ends. Possible cleavage permutations are shown diagrammatically in Fig. 10. The isoforms predicted by this model are almost identical in size to some of the isoforms identified in transfected Cos-7 cells (Fig. 3A), which strongly supports this model of protein processing. Previous studies have highlighted the importance of PEST domain exposure (ODC) (20, 28) and removal (NF-B) (30, 31) on degradation function. It is interesting therefore that we propose a pattern of processing for HDC in which both steps feature in regulation. This regulation could occur as part of either the degradation or activation pathway. While future studies will need to address the relative enzyme activities of isoforms generated by this hypothesized cleavage scheme, it is noteworthy that previous in vitro translation studies suggest that a 48-kDa core protein located between PEST1 and PEST2 is highly enzymatically active (15); however, this is the first study to show that generation of such an isoform might have any physiological relevance. While previous studies suggest that the production of smaller HDC isoforms requires the initial translation of the 74-kDa isoform (44), it is noteworthy that our results do not completely rule out the use of alternative translational start sites in the generation of multiple HDC isoforms.

View larger version (16K): [in this window] [in a new window]   FIG. 10.   Diagrammatic representation of hypothesized cleavage pattern of HDC, based on inclusion or exclusion of endoplasmic reticulum targeting and PEST domains. Numbered amino acids from the primary rat HDC protein sequence are shown underneath. The approximate locations of the antigenic determinants used in the generation of the anti-HDC antibody used in this study are shown by solid triangles above the sequence.

The presence of targeting and degradation elements (ER1/PEST1 and ER2/PEST2) (Fig. 10) at both ends of the enzyme raises some interesting questions regarding the relevance of successive processing steps on HDC function. One possibility is that the 74-kDa HDC is initially targeted to the endoplasmic reticulum for degradation. Under certain conditions, partial degradation of the 74-kDa isoform to remove ER2 could allow retrograde translocation and amino-terminal cleavage of a more stable (and consequently more active) isoform back to the cytosol, where it is believed that histamine is produced (3, 4). Such retrograde translocation to the cytosol is an accepted model of intracellular protein trafficking (50) and could result in degradation regulated by PEST1, which is shown here to be less susceptible to proteasome degradation than PEST2.

It is in this context, therefore, where one or more functional domains are likely to have been exposed or removed, that we need to consider gastrin regulation of HDC. Pulse-chase experiments demonstrated that gastrin disproportionally increases the turnover rates of the different HDC isoforms. We propose that this form of regulation depends on the presence or absence of amino- and carboxy-terminal PEST domains, which differentially stabilize heterologous proteins in response to gastrin stimulation. In this model, the expression of isoforms that lack PEST domains should also increase due both to stabilization of precursor isoforms and to slower rates of degradation. This model assumes a constant rate of conversion between isoforms and the potential for independent degradation of each of the isoforms. At the present time, however, we cannot determine categorically whether selective conversion of isoforms occurs or whether parameters known to regulate stability (such as covalent modification or dimerization [2, 36, 17]) might represent an additional level of regulation.

In our experiments we observed that the gastrin effect was cycloheximide sensitive. While this could imply the translation of factors that interact with specific HDC domains to regulate stability, our experiments showed that gastrin can affect the expression of heterologous proteins containing PEST domains from a variety of different sources, including ODC and, to a lesser extent, DDC. This suggests a more general effect on the turnover of PEST-containing proteins. In preliminary competition cotransfection experiments performed with pEP-HDC2.4 and increasing amounts of pBF-PEST2, we found no effect on basal HDC enzyme activity (data not shown), which further argues against specific factors interacting with the PEST2 domain to specifically regulate HDC degradation. Our results therefore suggest that gastrin stabilizes HDC isoforms by regulating factors common to the degradation of a number of rapidly turned-over proteins. This interpretation is consistent with experiments showing that the heterologous proteins most susceptible to gastrin stimulation were also the proteins most affected by lactacystin inhibition of the proteasome (BFP-PEST2 and GFP-PEST/ODC). This type of regulation could involve the gastrin-stimulated translation of one or more inhibitors of degradation, although such a pattern of regulation has not, to our knowledge, been described previously. Such regulation could have important implications for other proteins as well. For example, transcription factors (such as Fos and NF-B [35, 31]) and cell cycle regulators (such as cyclin D1 and cyclin G [35]) are also known to contain PEST domains. It is possible that gastrin could affect the steady-state levels of these proteins; indeed, such stabilization could contribute to the documented trophic effect of gastrin on ECL cells (38).

Interestingly, our results have identified an additional level at which ODC stability can be regulated. The GFP-PEST/ODC chimera used in these experiments lacks the antizyme-binding domain (27). Therefore, even though antizyme is required for the exposure of the carboxy-terminal PEST domain during normal degradation of the native ODC protein (14, 20, 28), our results indicate that there are additional factors, such as gastrin, which can regulate stability at points thereafter. Taken together with studies that demonstrate a role for gastrin response elements in transcriptional regulation (34, 49) and gastrin stimulation in the regulation of mRNA translation (33), our studies here confirm and extend evidence for multilayered control of gene expression by stimulation of G protein-coupled receptors. They also revealed a complex pattern of posttranslational regulation, with activity and stability determined by the presence or absence (through cleavage) of functional domains located within the tertiary protein sequence. Gastrin acts with an unidentified factor(s) to increase the half-lives of specific HDC isoforms. Extrapolation of these results suggests that increased histamine production, observed immediately after gastrin stimulation of ECL cells, occurs as a consequence of stabilization of HDC isoforms.