Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.     

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

Background  

Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.     

Cellular localization and developmental changes of the different isoforms of divalent metal transporter 1 (DMT1) in the inner ear of rats

Divalent metal transporter 1 (DMT1) is generally considered to be the major transmembrane protein responsible for the uptake of a variety of divalent cations. Four isoforms of DMT1 have been identified in mammalian cells encoded by a single gene that differ both in their N- and C-terminal sequences with two mRNA isoforms possessing an iron response element (IRE) motif downstream from the stop codon on the message. Two distinct promoter sites regulate production of the 1A or 1B isoforms (translation starts at exon 2) for both the +IRE or –IRE species of the transporter resulting in the generation of four distinct configurations of this protein. Prior studies from our laboratory using cochlear organotypic cultures isolated from postnatal day three rats (P3) have demonstrated that Mn causes significant and selective damage to sensory hair cells and auditory nerve fibers and spiral ganglion neurons in a time and concentration dependent manner. Since DMT1 plays a critical role in controlling the uptake of a variety of essential and toxic metals into the cochlea, we compared the distribution and developmental changes of the 1A, +IRE and ?IRE isoforms in rat inner ear. Results reveal that all three isoforms of DMT1 are selectively expressed in different cell populations within the cochlea and, additionally, demonstrate their cellular and subcellular distribution changes with development.     

Glycine transporter isoforms show differential subcellular localization in PC12 cells

The subcellular localization of glycine transporters one (GLYT1) and two (GLYT2) stably expressed in PC12 cells has been studied. To facilitate visualization, enhanced green fluorescent protein (GFP) was fused to the amino terminus of both glycine transporters. Functional analysis of the GFP-GLYT1 and GFP-GLYT2 stable cell lines demonstrated that they exhibited high affinity for glycine and the characteristic properties of both glycine transporter subtypes. The GFP-coupled transporters were differently distributed throughout the cell. GFP-GLYT1 was mainly localized on the plasma membrane, whereas most of GFP-GLYT2 was present on large dense-core vesicles and endosomes. Both transporters were absent from the synaptic vesicle population in PC12 cells.     

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.     

Alternative splicing regulates the endoplasmic reticulum localization or secretion of soluble secreted endopeptidase

A subfamily of zinc metalloproteases, represented by Neutral endopeptidase (EC ) and endothelin-converting enzyme, is involved in the metabolism of a variety of biologically active peptides. Recently, we cloned and characterized a novel member of this metalloprotease family termed soluble secreted endopeptidase (SEP), which hydrolyzes many vasoactive peptides. Here we report that alternative splicing of the mouse SEP gene generates two polypeptides, SEP(Delta) and SEP. After synthesis, both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. SEP(Delta) then becomes an ER resident, whereas SEP, which differs by only the presence of 23 residues at the beginning of its luminal domain, is proteolytically cleaved by membrane secretase(s) in the ER and transported into the extracellular compartment. An analysis of the chimeric proteins between SEP(Delta) and bovine endothelin-converting enzyme-1b (bECE-1b) demonstrated that the retention of SEP(Delta) in the ER is mediated by the luminal domain. In addition, the dissection of the chimeric bECE-1b/SEP insertion showed that its insertion domain is obviously responsible for its secretion. A series of mutagenesis in this region revealed that the minimal requirement for cleavage was found to be a WDERTVV motif. Our results suggest that the unique subcellular localization and secretion of SEP proteins provide a novel model of protein trafficking within the secretory pathway.     

Deficiency in the divalent metal transporter 1 increases bleomycin-induced lung injury

Exposure to bleomycin can result in an inflammatory lung injury. The biological effect of this anti-neoplastic agent is dependent on its coordination of iron with subsequent oxidant generation. In lung cells, divalent metal transporter 1 (DMT1) can participate in metal transport resulting in control of an oxidative stress and tissue damage. We tested the postulate that metal import by DMT1 would participate in preventing lung injury after exposure to bleomycin. Microcytic anemia (mk/mk) mice defective in DMT1 and wild-type mice were exposed to either bleomycin or saline via intratracheal instillation and the resultant lung injury was compared. Twenty-four h after instillation, the number of neutrophils and protein concentrations after bleomycin exposure were significantly elevated in the mk/mk mice relative to the wild-type mice. Similarly, levels of a pro-inflammatory mediator were significantly increased in the mk/mk mice relative to wild-type mice following bleomycin instillation. Relative to wild-type mice, mk/mk mice demonstrated lower non-heme iron concentrations in the lung, liver, spleen, and splenic, peritoneal, and liver macrophages. In contrast, levels of this metal were elevated in alveolar macrophages from mk/mk mice. We conclude that DMT1 participates in the inflammatory lung injury after bleomycin with mk/mk mice having increased inflammation and damage following exposure. This finding supports the hypothesis that DMT1 takes part in iron detoxification and homeostasis in the lung.     

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. ⁶⁴Cu¹⁺ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. ⁶⁴Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein^?1 min^?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu¹⁺.     

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.