Amino- and carboxy-terminal PEST domains mediate gastrin stabilization of rat L-histidine decarboxylase isoforms.

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.