A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.     

Beta spectrin in human skeletal muscle. Tissue-specific differential processing of 3' beta spectrin pre-mRNA generates a beta spectrin isoform with a unique carboxyl terminus

Spectrin, an important component of the mammalian erythrocyte membrane skeleton, is a heterodimeric protein with alpha and beta subunits of 280 and 246 kDa, respectively. Spectrin-like proteins have also been demonstrated in a wide variety of nonerythroid cells. To examine the hypothesis that nonerythroid beta spectrins may be encoded by the "erythroid" beta spectrin gene, we have isolated cDNA clones from a human fetal skeletal muscle library by hybridization to a previously described red cell beta spectrin cDNA. Detailed comparison of muscle and erythroid beta spectrin cDNAs has revealed sequence identity over the majority of their lengths, confirming that they are the product of the same gene. However, there is a sharp divergence in sequence at their 3' ends. A consequence of this divergence is the replacement of the carboxyl terminus of erythroid beta spectrin with a different, longer carboxyl-terminal domain in skeletal muscle. We hypothesize that tissue-specific differential polyadenylation leads to the selective activation of a donor splice site within the beta spectrin coding sequence, splicing downstream nonerythroid exons into the mature muscle beta spectrin mRNA. We predict that replacement, in nonerythroid cells, of the beta spectrin carboxyl terminus, known to participate in spectrin self-association and phosphorylation, has significant functional consequences. These data may explain previously reported nonerythroid beta spectrin isoforms that resemble red cell beta spectrin by immunochemical analysis.     

O-N-acetyl-D-glucosamine moiety on discrete peptide of multiple protein 4.1 isoforms regulated by alternative pathways

Erythrocyte protein 4.1 is a cytoplasmic protein that possesses a protein-saccharide modification structure, an O-N-acetyl-D-glucosamine (GlcNAc) moiety. We determined the amino acid sequence of the proteolytic fragment containing the O-GlcNAc moiety after labeling the saccharide with [3H]galactose in the presence of bovine milk galactosyltransferase. Glycosylation appears to occur on one or more serine or threonine residues in the following sequence: Thr-Ala-Gln-Thr-Ile-Thr-Ser-Glu-Thr-Pro-Ser-Ser-Thr-Thr-Thr-Thr-Gln-Ile-Thr-Lys . This sequence corresponds to the carboxyl-terminal half of the 34-amino acid peptide in the 22/24-kDa carboxyl-terminal domain of protein 4.1, which is one of the discrete peptides regulated by alternative RNA splicing. Multiple protein 4.1 isoforms in erythroid and nonerythroid cells including major components of erythrocyte membrane proteins, 4.1a and 4.1b, appear to contain this sequence since most immunochemically reactive proteins were labeled with [3H]galactose, with the exception of several variant polypeptides. These results appear to suggest the functional or biological significance of the O-GlcNAc linkage in protein 4.1.     

Organization of the Human Protein 4.1 Genomic Locus: New Insights into the Tissue-Specific Alternative Splicing of the Pre-mRNA

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin–actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones 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, accounts for one-half of the mature mRNA.     

Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1

Protein 4.1, a multifunctional structural protein originally described as an 80-kDa component of the erythroid membrane skeleton, exhibits tissue- and development-specific heterogeneity in molecular weight, subcellular localization, and primary amino acid sequence. Earlier reports suggested that some of this impressive heterogeneity is generated by alternative RNA splicing (Conboy, J. G., Chan, J., Mohandas, N., and Kan, Y. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9062-9065; Tang, T. K., Leto, T., Marchesi, V. T., and Benz, E. J. (1990) J. Cell Biol. 110, 617-624). We have now completed a systematic analysis of 4.1 mRNA isoforms expressed in erythroid cells, and have generated an "alternative splicing map" which summarizes diagrammatically a multitude of polypeptide isoforms potentially generated by combinatorial splicing of nine alternative exons. Complex 5' splicing events yield mRNA isoforms that may initiate translation at different sites and thus generate elongated or truncated NH2 termini; elongated approximately 135-kDa and prototypical approximately 80-kDa species were detected in both erythrocytes and T-lymphocytes, but in very different ratios. Among the functional domains of 4.1 responsible for interaction with other membrane skeletal elements, four variants of the 10-kDa spectrin-actin-binding region and four variants of the putative 30-kDa glycophorin-binding region are predicted. Developmentally controlled alternative RNA splicing in the spectrin-actin-binding region may help regulate remodeling of membrane architecture and mechanical properties that occur during erythropoiesis.     

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.     

An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

Background  

In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.     

An analogue of the erythroid membrane skeletal protein 4.1 in nonerythroid cells

Protein 4.1 is a crucial component of the erythrocyte membrane skeleton. Responsible for the amplification of the spectrin-actin interaction, its presence is required for the maintenance of erythrocyte integrity. We have demonstrated a 4.1-like protein in nonerythroid cells. An antibody was raised to erythrocyte protein 4.1 purified by KCl extraction (Tyler, J. M., W. R. Hargreaves, and D. Branton, 1979, Proc. Natl. Acad. Sci. USA, 76:5192-5196), and used to identify a serologically cross-reactive protein in polymorphonuclear leukocytes, platelets, and lymphoid cells. The cross-reactive protein(s) were localized to various regions of the cells by immunofluorescence microscopy. Quantitative adsorption studies indicated that at least 30-60% of the anti-4.1 antibodies reacted with this protein, demonstrating significant homology between the erythroid and nonerythroid species. A homologous peptide doublet was observed on immunopeptide maps, although there was not complete identity between the two proteins. When compared with erythrocyte protein 4.1, the nonerythroid protein(s) displayed a lower molecular weight--68,000 as compared with 78,000-and did not bind spectrin or the nonerythroid actin-binding protein filamin. There was no detectable cross-reactivity between human acumentin or human tropomyosin-binding protein, which are similarly sized actin-associated proteins, and erythrocyte protein 4.1. The possible origin and significance of 4.1-related protein(s) in nonerythroid cells are discussed.     

Tissue-specific analogues of erythrocyte protein 4.1 retain functional domains

Analogues of the human erythroid membrane skeletal component protein 4.1 have been identified in perfused rat tissues and human T and B lymphocyte cell lines. olyclonal antibodies were used which are specific for all domains of protein 4.1, the spectrin-actin-promoting 8-Kd peptide, the membrane-binding 30-Kd domain, and the 50-Kd domain. Antibody reactivity, by Western blotting of tissue homogenates, shows reactivity with proteins varying in molecular weight from 175 Kd to 30 Kd. Further, these protein 4.1 analogues appear to be expressed in a tissue-specific fashion. Of the analogues detected there appear to be at least three classes: analogues containing all erythroid protein 4.1 domains, analogues containing all domains but with modified antigenic epitopes, and analogues containing only some domains. Chemical cleavage at cysteine linkages indicates that in analogues containing the 30-Kd region the location of cysteine is highly conserved. This datum suggests that in nonerythroid 4.1 isoforms of higher molecular weight the additional protein mass is added to the amino terminal end (30 Kd end).