Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage

Multiple endocrine neoplasis type 2A (MEN2A) is one of several kinds of cancers that appear to be inherited in an autosomally dominant fashion. We have assigned the MEN2A locus to chromosome 10 by linkage with a new DNA marker (D10S5). The linkage led us to investigate other chromosome 10 markers and demonstrate linkage between the disease locus and the interstitial retinol-binding protein (IRBP) gene. The D10S5 locus was sublocalized to 10q21.1 by hybridization in situ and the IRBP gene to p11.2----q11.2 with a secondary site at q24----q25. The linkages were established using 292 members of five families, three different restriction fragment length polymorphisms (RFLPs) at D10S5 and two RFLPs recognized by the IRBP probe. The recombination frequencies from pairwise linkage analysis between the disease and two marker loci D10S5 and IRBP were 0.19 and 0.11, with maximum lod scores of 3.6 and 8.0 respectively. Ordering of the three loci by multipoint analysis placed the IRBP gene approximately midway between the disease and D10S5 loci.     

Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.     

Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer

Specific chromosomal deletions sometimes associated with tumours such as retinoblastoma (chromosome 13q14) and Wilm's tumour (chromosome 11p13) have led to the hypothesis that recessive genes may be involved in tumorigenesis. This hypothesis is supported by demonstration of allele loss specific for these regions using polymorphic DNA markers and by the isolation of a complementary DNA clone for the retinoblastoma gene. A cytogenetic deletion in chromosome 3 (p14-p23) was reported in small-cell lung cancer (SCLC) by Whang-Peng et al. At least one homologue of chromosome 3 was affected in the majority of SCLC tumours; however, the multiple chromosomal changes seen presented the possibility that chromosome 3 was rearranged, not deleted. We used polymorphic DNA probes for chromosome 3p and compared tumour and constitutional genotypes of nine SCLC patients. Our data show loss of alleles of chromosome 3p markers in tumour DNA of all nine patients supporting the hypothesis that this region contributes to tumorigenesis in SCLC.     

Development of homozygosity for chromosome 11p markers in Wilms' tumour

Somatic alterations in the genome are found in many human tumours. Chromosome rearrangements or base substitutions that activate cellular oncogenes appear to act dominantly. In contrast, recessive alleles apparently contribute to childhood retinoblastoma, as homozygosity (or hemizygosity ) for chromosome 13 is often established in tumours, by either mitotic nondisjunction or recombination. Parallels exist between retinoblastoma and childhood Wilms' tumour (WT). Retinoblastoma is often inherited and accompanied by a deletion of chromosome 13 (band q14), while WT is occasionally associated with aniridia and deletion of chromosome 11 band p13. Most Wilms' tumours are sporadic and not accompanied by these findings, although interstitial deletion of chromosome 11 in tumour, but not normal, cells has been reported. In view of these parallels, we compared constitutional and tumour DNAs from WT patients by using chromosome 11p DNA probes. We report here that although heterozygosity in constitutional DNAs was often preserved in tumour DNAs, one case developed homozygosity for chromosome 11p markers in tumour cells, implying the involvement of chromosomal events in revealing a recessive WT locus. This observation suggests the action of such general mechanisms in a tumour other than retinoblastoma.     

Loss of alleles at loci on human chromosome 11 during genesis of Wilms' tumour

Evidence that recessive cellular alleles at specific chromosomal loci are involved in tumorigenesis has been recently shown by work on tissues from patients with retinoblastoma, a neoplasm of embryonic retina whose predisposition is inherited as an autosomal dominant trait. A comparison of germ-line and tumour genotypes at loci on human chromosome 13, defined by restriction fragment length polymorphisms, showed that loss of the chromosome bearing the wild-type allele at the Rb-1 locus occurred frequently in the development of retinoblastoma. We report here results of similar studies of another embryonal neoplasm, Wilms' tumour of the kidney. Examination of germ-line and tumour genotypes from seven patients showed that five cases were consistent with the presence on human chromosome 11 of a locus in which recessive mutational events are expressed after abnormal chromosomal segregation events during mitosis.     

Genetic homogeneity between acute and chronic forms of spinal muscular atrophy

The childhood-onset spinal muscular atrophies (SMAs) describe a heterogeneous group of disorders that selectively affect the alpha motoneuron. We have shown that chronic childhood-onset SMA (SMA II and III) maps to a single locus on chromosome 5q. Acute SMA (SMA Type I/Werdnig-Hoffmann/severe/infantile) is the main cause of heritable infant mortality. Mapping the acute SMA locus by conventional methods is complicated by the rapidly fatal course of the disease and its recessive mode of inheritance. We present here the typing of four inbred acute-SMA families with DNA markers on chromosome 5q and analysis of these together with acute families from our previous study to demonstrate genetic homogeneity between the acute and chronic forms of SMA. The data indicate that the acute SMA locus maps to chromosome 5q11.2-13.3. Two families seem unlinked to 5q markers, raising the possibility of genetic heterogeneity or disease misclassification within the acute and chronic family sets.     

Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation

The t(11;14) (q13;q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL) and multiple myeloma. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8;14) (q24;q32) translocation of the Burkitt tumour, interruption of the immunoglobulin heavy-chain locus was postulated for this rearrangement. We have cloned the chromosomal joinings between chromosomes 11 and 14 and also between chromosomes 14 and 18, in B-cell tumours carrying translocations involving these chromosomes, and suggested the existence of two translocated loci, bcl-1 and bcl-2, normally located on chromosomes 11 (band q13) and 18 (band q21) respectively, involved in the pathogenesis of human B-cell neoplasms. The results indicate that in the leukaemic cells from two different cases of CLL, the breakpoints on chromosome 11 are within 8 nucleotides of each other and on chromosome 14 involve the J4-DNA segment. Because we detected a 7mer-9mer signal-like sequence with a 12-base-long spacer on the normal chromosome 11, close to the breakpoint, we speculate that the t(11;14) chromosome translocation in CLL may be sequence specific and may involve the recombination system for immunoglobulin gene segment (V-D-J) joining.     

Partial deficiency of erythrocyte spectrin in hereditary spherocytosis

Hereditary spherocytosis (HS) is a common, clinically heterogeneous haemolytic anaemia in which the primary erythrocyte defect is believed to be some abnormality in the spectrin-actin membrane skeleton, leading to loss of surface membrane. Recessively inherited spectrin deficiency with extreme erythrocyte fragility and spherocytosis has been identified in certain mutant mice and two severely anaemic humans. Although suspected, deficiency of spectrin has not been demonstrated in less severe forms of human HS. We not report the quantitation of erythrocytes spectrin by radioimmunoassay. We found that normal erythrocytes contained 240,000 copies of spectrin heterodimer, whereas erythrocytes from 14 patients with a variety of types of HS were all partially deficient in spectrin (range 74,000-200,000 copies), the magnitude of the deficiency correlating with the severity of the disease. Spectrin deficiency of varying degrees is common in HS and probably represents the principal structural defect leading to loss of surface membrane.     

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.     

Loss of constitutional heterozygosity in colon carcinoma from patients with familial polyposis coli

Recent studies have suggested a critical role of specific gene loss in several embryonic tumours and certain adult cancers. In retinoblastoma, hemizygosity or homozygosity of a recessive mutant allele results in the loss of normal gene product, and this seems to cause the manifestation of the disorder. Familial polyposis coli (FPC) is a human autosomal dominant trait characterized by numerous adenomatous polyps of the colon and rectum, and a high incidence of colon carcinoma. Karyotype analyses have failed to detect specific deletion or translocation. We report the use of polymorphic DNA markers to look for the somatic loss of heterozygosity at specific loci. Investigation of 38 tumours from 25 FPC patients, and 20 sporadic colon carcinomas from 19 patients, revealed frequent occurrence of allele loss on chromosome 22, with some additional losses on chromosomes 5, 6, 12q and 15. The FPC gene-linked DNA probe C11p11 also detected frequent allele loss in both familial and sporadic colon carcinomas but not in benign adenomas. These results suggest the possible involvement of more than one chromosomal locus in the development of familial and sporadic colon carcinomas.     

Loss of genes on the short arm of chromosome 11 in bladder cancer

Recent studies have shown that normal cellular sequences on chromosome 13 are lost during the development of retinoblastomas and that sequences on chromosome 11 are similarly lost during the development of Wilms' kidney tumours and embryonal tumours. Cells from these tumors have been found to contain either the paternal or maternal copies of loci on the affected chromosome, but not both. Thus, the somatic loss of heterozygosity for sequences on chromosome 13 or 11 is hypothesized to result in homozygosity for a recessive mutant allele on these chromosomes, and in this way the chromosomal loss may contribute to the development of these tumours. We sought to investigate whether similar losses of heterozygosity for chromosome 11 sequences occurred in a common adult tumour. We chose to analyse bladder cancers, since such cancers are common in the adult population and are derived from urogenital tissue, as are Wilms' tumours. We examined constitutional and tumour genotypes at loci on the short arm of chromosome 11 (11p) in 12 patients with transitional cell carcinomas. In five tumours, we observed the somatic loss of genes on 11p resulting in homozygosity or hemizygosity of the non-deleted alleles in the tumour cells. Our results show that the frequency of loss of 11p sequences in bladder cancer approaches that seen in Wilms' tumour (42% compared with 55%), and suggest that recessive genetic changes involving sequences on 11p may contribute to the development of bladder neoplasms.     

Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide

Many of the physical properties of the erythrocyte membrane appear to depend on the membrane skeleton, which is attached to the membrane through associations with transmembrane proteins. A membrane skeletal protein, protein 4.1, is pivotal in the assembly of the membrane skeleton because of its ability to promote associations between spectrin and actin. Protein 4.1 also binds to the membrane through at least two sites: a high-affinity site on the glycophorins and a site of lower affinity associated with band 3 (ref. 11). The glycophorin-protein 4.1 association has been proposed to be involved in maintenance of cell shape. Here we show that the association between glycophorin and protein 4.1 is regulated by a polyphosphoinositide cofactor. This observation suggests a mechanism which may explain the recently reported dependence of red cell shape on the level of polyphosphoinositides in the membrane.     

Spectrin assembly in avian erythroid development is determined by competing reactions of subunit homo- and hetero-oligomerization

Erythroid differentiation entails the biogenesis of a membrane skeleton, a network of proteins underlying and interacting with the plasma membrane, whose major constituent is the heterodimeric protein spectrin, composed of two structurally similar but distinct subunits, alpha (relative molecular mass (Mr) 240,000) and beta (Mr 220,000), which interact side-on with each other to form a long rod-like molecule. Interaction of this network with the membrane is mediated by the binding of the beta subunit to ankyrin, which in turn binds to the cytoplasmic domain of the transmembrane anion transporter (also referred to as band 3). Purified alpha and beta subunits of spectrin from the membrane of mature red blood cells will spontaneously heterodimerize, suggesting that assembly of the spectrin-actin skeleton is a simple self-assembly process, but in vivo studies with developing chicken embryo erythroid cells have indicated that assembly in vivo is more complex. We now present evidence that newly synthesized spectrin subunits in vivo or in vitro rapidly adopt one of two competing conformations, a heterodimer or a homo-oligomer. These competing reactions seem to determine the overall extent of spectrin assembled during erythroid development by determining which conformation will assemble onto the membrane-skeleton (the heterodimer) and which conformations are targeted for degradation (the homo-oligomers).     

Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins

Analysis of complementary DNA for human erythroid ankyrin indicates that the mature protein contains 1,880 amino acids comprising an N-terminal domain binding integral membrane proteins and tubulin, a central domain binding spectrin and vimentin, and an acidic C-terminal 'regulatory' domain containing an alternatively spliced sequence missing from ankyrin variant 2.2. The N-terminal domain is almost entirely composed of 22 tandem 33-amino-acid repeats. Similar repeats are found in yeast and invertebrate proteins involved in cell-cycle control and tissue differentiation.