Domain structure and transcript diversity of plectin.
We have cloned and sequenced plectin from rat (Wiche et al., 1991) and man (Liu et al., 1996). Secondary structure predictions based on the deduced amino acid sequences of cDNAs and genomic clones, as well as on electron microscopy of the protein (Foisner and Wiche, 1987), revealed a multi-domain structure composed of a central [approximately]200 nm long, [Alpha]-helical coiled-coil structure flanked by large globular domains. The structure of the carboxy-terminal domain is dominated by six highly homologous repeats that also occur in lesser number in desmoplakin (3 repeats; Green et al., 1990), bullous pemphigoid antigen (BPAG) 1 (2 repeats; Sawamura et al., 1991), and the recently identified envoplakin (1 repeat; Ruhrberg et al., 1996). Analysis of the human gene locus revealed a complex organization of 32 exons spanning 31 kb of DNA located in the telomeric region (q24) of chromosome 8 (Liu et al., 1996).
On the molecular level, plectin binds to a variety of cytoskeletal proteins, including cytoplasmic and nuclear IF subunit proteins (vimentin, GFAP, cytokeratins, neuro-filament proteins, lamin B), subplasma membrane proteins (fodrin and [Alpha]-spectrin), and high molecular weight microtubule-associated proteins MAP1 and MAP2 (Herrmann and Wiche, 1987; Foisner et al., 1988; Wiche et al., 1993). The expression of mutant forms of plectin in cell lines transiently transfected with cDNA constructs led to the conclusion that the C-terminal globular domain of plectin is involved in the binding to IFs (Foisner et al., 1991). Recently, we mapped the binding site of plectin to vimentin, and to keratinocyte cytokeratins, to a stretch of [approximately]50 amino acid residues within plectin's terminal repeat 5-domain; and a basic amino acid residue cluster within a functional nuclear targeting sequence motif was identified as an essential element of this site (Nikolic et al., 1996). Moreover, we found that a dystrophin/[Beta]spectrin-like, actin-binding domain of plectin - located in its aminoterminal region (encoded by exons 2-8) - is functional.
Plectin is characterized by versatile binding activities, prominence at distinct strategically important locations within the cytoarchitecture (such as cytoskeleton anchorage junctions), complex exon-intron organization, and differential staining of tissues and cells as revealed by a battery of monoclonal antibodies raised to the protein. These features suggest that different plectin isoforms exist, that they perform different cellular tasks and, thus, have different subcellular localizations. Recently, we have found several such variant transcripts in rat and man. Of particular interest was the identification of four distinct first coding exons, all of which splice into a common successive exon 2. RNase protection mapping of transcripts containing three of the four identified alternative first exons revealed their coexpression in rat glioma C6 cells, and in a series of different rat tissues. However, significant variations in the expression levels of first exons indicated tissue-specific promoters for at least some of them. Multiple transcriptional start sites and a preceding, nontranscribed GC-rich sequence lacking any TATA element suggested that expression of exon 1 transcripts involves a promoter characteristic of housekeeping genes. In addition, plectin splice variants lacking exon 31 ([greater than]3 kb), which encodes the entire rod domain of the molecule, were identified by RT-PCR in a variety of cells and tissues. These findings lend further support to the hypothesis that plectin is a versatile organizing element of the cytoskeleton, and they provide first insights into a complex gene regulatory machinery.
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LUNA: You said your plectin knockout mice had a severe phenotype; would you elaborate on that?
WICHE: They have a severe phenotype, with skin blistering from which they die within two days after birth. We are looking to see if they show additional phenotypes.
ALBRECHT-BUEHLER: Plectin seems to be a dangerous molecule for a cell to have. As you described it, it acts basically like a fixative, so a cell must control this molecule if it wants to live. The isoforms you describe are one of the possible ways for taking advantage of the multivalency of such a fixative. I would expect that there is much more to it. Is there any evidence for some kind of chaperone, some kind of sequestering proteins, that deliver plectin to places rather than letting it freely fix any protein that it meets? Have you ever looked for something like this?
WICHE: No we haven't.
BORISY: You talked about all of the different molecules that plectin interacts with, and then you told us about the possibility of many plectin isoforms. Do you think that individual plectin molecules can bind to all of the different targets, or do you think that the spectrum of binding of a particular isoform is more limited, with some of the multiplicity of binding being due to different isoforms?
WICHE: Yes, I would predict that the spectrum of binding of the molecule is limited. Different molecules have different tasks; one molecule cannot do everything. By forming complexes, even by dimerization of different isoforms, different functional properties of the formed molecule may be obtained.
BORISY: A second question is, if intermediate filament proteins are the major binding partner for plectins, how do you explain the phenotype of the vimentin knockout mouse reported in the literature?
WICHE: I wouldn't say that intermediate filaments are necessarily the major interaction partner. That was the conclusion based on early experiments, where we found and isolated isoforms of plectin due to their binding to intermediate filaments. When we characterize more of these forms, this picture will probably change. Regarding the vimentin knockout mouse, perhaps plectin is more important than the vimentin in this. We have studied plectin expression and organization in the cytoplasm of vimentin-negative cells. Plectin is there as a network, even without vimentin.
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|Title Annotation:||The Cytoskeleton: Mechanical, Physical, and Biological Interactions; includes panel discussion|
|Publication:||The Biological Bulletin|
|Date:||Jun 1, 1998|
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