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EVOLUTIONARY HOMOLOGS (part 1/2)

Protein 4.1 homologs

Another Drosophila protein with homology to protein 4.1 is the recently discovered Inscuteable (Kraut, 1996).

The Drosophila expanded (ex) gene encodes a product (Ex) that shares homology with the family of protein 4.1 proteins, many of which are found localized at specific lateral cell junctions and the apical cellular domain. Ex colocalizes with actin in the apical domain of imaginal disc epithelial cells, where it partially overlaps the distribution of proteins containing phosphotyrosine (PY). This suggests that Ex is present in or associated with adherens junctions. Genetic studies show that Ex is necessary for proper regulation of final cell number in adult wings and for the formation of eyes, distal leg, and distal antennal segments. Mitotic clones were generated that lack Ex, using the twin spot technique. These clones demonstrate that the primary function of Ex is to regulate cell proliferation. Overexpressing Ex protein results in a decrease in final cell number in wings, suggesting a direct relationship between Ex function and proliferation rate (Boedigheimer, 1997).

A protein tyrosine phosphatase (PTP), designated PTPMEG, contains sequence homology to protein 4.1. Recombinant protein and amino- and carboxyl-terminal peptides were used to obtain polyclonal antibodies against PTPMEG to identify endogenous PTPMEG in A172 cells and to show that the enzyme is primarily localized to the membrane and cytoskeletal fractions of these cells. The protein is phosphorylated in both cell types on serine and threonine residues. The multiple sites of phosphorylation are all within the intermediate domain of the protein between amino acids 386 and 503. This region also contains two PEST sequences and two proline-rich motifs that may confer binding to Src homology 3 domains (Note: proteins with a domain known as the Src homology 3 domain are known to bind proline rich motifs). The recombinant protein is cleaved by trypsin and calpain in this region and thereby activated 4-8-fold. The full-length molecule is found in extracts from unstimulated platelets, whereas extracts from both calcium ionophore- and thrombin-treated platelets contain proteolyzed and activated forms of the enzyme, indicating that proteolysis by calpain is evoked in response to thrombin. Prior incubation of platelets with calpeptin, an inhibitor of calpain, blocks the agonist-induced proteolysis (Gu, 1996).

Mutation of protein 4.1

Multiple protein 4.1 isoforms are expressed in a variety of tissues through complex alternative pre-mRNA splicing events, one function of which is to regulate use of two alternative translation initiation signals. Late erythroid cells express mainly the downstream initiation site for synthesis of prototypical 80-kD isoforms; in addition, nonerythroid cells use an upstream site to encode higher molecular mass isoform(s). The effects of a 5' gene rearrangement were studied in a family with hereditary elliptocytosis and complete deficiency of erythrocyte 4.1 protein on 4.1 isoform expression in erythroid vs. nonerythroid cells. Patient 4.1 mRNAs from reticulocytes, fibroblasts, and B lymphocytes were amplified by reverse transcriptase/polymerase chain reaction techniques and shown to exhibit a 318-nucleotide deletion that encompasses the downstream AUG, but leaves intact the upstream AUG. Immunoblot analysis revealed a total deficiency of 4.1 in patient red cells and a selective deficiency of 80-kD isoform(s) but not high molecular weight 4.1 in patient nonerythroid cells. Thus, the 4.1 gene mutation in this family produces an isoform-specific deficiency that is manifested clinically in tissue-specific fashion, such that red cells are affected but other cell types are unaffected because of tissue-specific differences in RNA splicing and translation initiation (Conboy, 1993).

The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa, has been attributed to complex alternative splicing of the 4.1R gene. Two new 4.1 genes have been identified: one is generally expressed throughout the body (4.1G) and the other is expressed in central and peripheral neurons (4.1N). 4.1R is found to be selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R are discretely localized to granule cells in the cerebellum and dentate gyrus. Mice that lack 4.1R expression were generated; these mice have deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system (Walensky, 1998).

Multiple transcripts and splicing of protein 4.1

Expression of multiple protein 4.1 isoforms in erythroid progenitors and in a variety of nonerythroid tissues results from alternative pre-mRNA splicing. In 4.1 pre-mRNA, several translation initiation sites are present; synthesis of isoforms larger than 80 kD occurs when an upstream 5' AUG is spliced in, whereas the 80-kD mature erythroid isoform is produced when the upstream AUG is spliced out and translation is initiated at the downstream AUG. During erythropoiesis, this splicing switch is developmentally regulated. This developmental switch was studied in hereditary elliptocytosis 4.1Alg, in which a DNA rearrangement involving the exon containing the downstream AUG results in loss of coding capacity for the 80-kD 4.1, leading to mature red blood cells deficient in 4.1 with decreased membrane mechanical stability. Analysis of erythroblast RNA shows that although it retains the upstream AUG, its coding region is approximately 2.2 kb, compared with approximately 2.5 kb of normal 4.1 mRNA, because of the deletion of exons, including the one that codes for the downstream AUG. These studies emphasize the crucial role of differentiation-regulated RNA splicing: within the same erythroid tissue, the HE 4.1Alg phenotype does not appear until after the differentiation-associated splicing event (Chasis, 1996).

Protein 4.1 is a multifunctional protein with heterogeneity in molecular weight, intracellular localization, tissue- and development-specific expression patterns. Mouse protein 4.1 gene, over 90 kilobases long, comprises at least 23 exons (13 constitutive exons, 10 alternative exons) interrupted by 22 introns. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. No significant sequence difference has been observed between splice junctions of alternative and constitutive exons. Apparently, most alternative exon-encoded peptides are located within particular functional domains of protein 4.1: two peptides encoded by alternative exons 4 and 5 are located near or within the glycophorin/calmodulin binding domain, whereas three other alternative exon-encoded peptides (a stretch of 19-amino acids encoded by exon 14, one of 14-amino acids encoded by exon 15, and a 21-amino acid stretch encoded by exon 16) are located near or within the spectrin-actin binding domain (See Drosophila Spectrin). Selective use of exon 2', which carries an upstream translation initiation codon (AUG), may produce an elongated P4.1 isoform (135 kDa) that is predominantly expressed in nonerythroid tissues. Combinatorial splicing of these exons may generate different isoforms that will exhibit complicated tissue-specific expression patterns (Huang, 1993).

Both protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the spectrin-actin binding domain of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette were found to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette do not function in these assays. The 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 suggest different, yet to be defined functions (Discher, 1993).

Expression of the complex gene encoding multiple isoforms of structural protein 4.1 is regulated by alternative pre-mRNA splicing. During erythropoiesis, developmental stage-specific inclusion of exon 16 generates protein 4.1 isoforms having a fully functional spectrin-actin binding domain. Human mammary epithelial cells (HMEC), coincident with the dramatic morphological changes induced by altered culture conditions, exhibit a novel pre-mRNA splicing switch involving a new exon (exon 17B, 450 nucleotides) in the COOH-terminal coding region. 4.1 RNA expressed in proliferating HMEC adherent to culture dishes mostly excludes exon 17B, whereas 4.1 transcripts processed in nondividing suspension cultures of HMEC strongly include this exon. This pre-mRNA splicing switch is reversible: cells transferred from poly(2-hydroxyethyl methacrylate) back to plastic resume cell division and down-regulate exon 17B expression. More detailed studies reveal complex tissue-specific alternative splicing of exon 17B and another new exon 17A (51 nucleotides). These results predict the existence of multiple 4.1 protein isoforms with diverse COOH termini. Moreover, they strongly suggest that regulation of gene expression during differentiation of epithelial cells is mediated not only by transcriptional mechanisms, but also by post-transcriptional processes such as alternative pre-mRNA splicing (Schischmanoff, 1997).

By tissue screening for protein 4.1 isoforms, new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain have been observed. In particular, a new 51-nt exon was found that is present almost exclusively in muscle tissue. In addition, multiple genomic clones have been isolated spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3'-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3' UTR (over 3 kb in size) accounts for one-half of the mature mRNA (Baklouti, 1997).

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. Multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. For example, exon 16 encodes a 21-amino acid (21aa) segment located in the 10-kD "spectrin/actin binding domain" (SAB), the presence of which is required for junctional complex stability in RBCs. Among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. Only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5' end and thus generating a high molecular weigh isoform, is found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. These results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma (Baklouti, 1996).

Protein 4.1 interaction with vertebrate Discs large

Continued: see Evolutionary homologs part 2/2 |

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