Protein 4.1 interaction with vertebrate Discs large

A human B-lymphocyte 100-kDa protein shares 60% amino acid identity with Drosophila Discs large. This human homolog known as hDlg contains a C-terminal domain homologous to the known guanylate kinases, a Src homology 3 region motif, and three repeats known as the Dlg repeat after Drosophila Discs large. Two nonhomologous domains that can contain in-frame insertions result in at least four alternatively spliced isoforms of hDlg. Several hDlg RNA transcripts are widely distributed in human and murine tissues, and the protein is localized to regions of cell-cell contact. Protein 4.1, the defining member of a family that includes talin and merlin/schwannomin, has the same cellular localization as hDlg, and two sites within hDlg associate in vitro with the 30-kDa N-terminal domain of protein 4.1 (Lue, 1994).

hDlg, a human homolog of the Drosophila Dlg tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. The PDZ-protein 4.1 interaction has been further defined in vitro and the functional role of both 4.1 binding sites in situ is shown. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. Both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2-mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. These data elucidate the multivalent nature of membrane-associated guanylate kinase homolog (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function (Lue, 1996).

The human homolog (hDlg) of the Drosophila Discs large tumor suppressor (Dlg) is a multidomain protein consisting of a carboxyl-terminal guanylate kinase-like domain, an SH3 domain, and three slightly divergent copies of the PDZ (DHR/GLGF) domain. The structural organization of the three PDZ domains of hDlg were examined using a combination of protease digestion and in vitro binding measurements. The PDZ domains are organized into two conformationally stable modules: one (PDZ1+2), consists of PDZ domains 1 and 2, and the other (PDZ3) corresponds to the third PDZ domain. Using amino acid sequencing and mass spectrometry, the boundaries of the PDZ domains after digestion with endoproteinase Asp-N, trypsin, and alpha-chymotrypsin were determined. The purified PDZ1+2, but not the PDZ3 domain, contains a high affinity binding site for the cytoplasmic domain of Shaker-type K+ channels. Similarly, the PDZ1+2 domain can also specifically bind to ATP. The binding of ATP to the PDZ domains may have several important functional implications:

(1) in a subset of MAGUKs the guanylate kinase domain shows a defective ATP-binding site. The binding of ATP to PDZ domains may bring an ATP molecule in proximity to the guanylate kinase domain, thus making catalysis possible.

(2) the hydrolysis of bound ATP may also induce conformational changes in MAGUKs.

(3) binding of ATP by PDZ domains may indirectly affect the functions of other proteins with which MAGUKs interact. An in vivo interaction occurs between hDIg and protein 4.1 and the hDIg protein contains a single high affinity protein 4.1-binding site that is not located within the PDZ domains.

The results suggest a mechanism by which PDZ domain-binding proteins may be coupled to ATP and the membrane cytoskeleton via hDlg (Marfatia, 1996).

Protein 4.1 interaction with actin

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high-affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Tissue-specific alternative splicing of exon 16 also occurs in frogs. Xenopus erythroid 4.1 demonstrates specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison shows two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Early embryos show high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms that are functionally competent for spectrin-actin binding (Winardi, 1995).

The spectrin-actin-binding domain of protein 4.1 is encoded by a 21-amino acid alternative exon and a 59-amino acid constitutive exon. To characterize the minimal domain active for interactions with spectrin and actin, recombinant 4.1 peptides containing the 21-amino acid cassette plus varying portions of the 59-amino acid cassette (designated 21.10 to 21.59) were functionally characterized. Peptide 21.43 is fully functional in binary interactions with spectrin and in ternary complex formation with spectrin and actin. Further truncation produces peptides incapable of binary interactions but fully competent for ternary complex formation (peptides 21.36 and 21.31), shorter peptides with reduced ternary complex activity and altered kinetics (21.26 and 0.59), and inactive peptides (21.20 and 21.10). Experiments suggest that residues 37-43 of the constitutive domain are directly involved in spectrin binding. These data indicate that 4.1-spectrin binary interaction requires the 21-amino acid alternative cassette plus the 43 N-terminal residues of the constitutive domain. The existence of two possible ternary complex assembly pathways is suggested: one initiated by 4.1-spectrin interactions, and a second by 4.1-actin interactions. The latter may require a putative actin binding motif within the 26 N-terminal residues of the constitutive domain (Schischmanoff, 1995).

The binary interaction between human erythrocyte protein 4.1 and rabbit skeletal muscle F-actin was examined by rapid pelleting of the binary complexes. The reaction is saturable at approximately one protein 4.1 molecule/actin monomer and is highly co-operative. The rate of reaction between protein 4.1 and actin is temperature sensitive in a manner consistent with a high energy of activation. Polyvalent anions disrupt the binary interaction between F-actin and protein 4.1. It is postulated that the co-operativity of the binding of protein 4.1 to actin results from a protein 4.1 molecule binding to monomers within the actin filament structure that then promotes conformational changes allowing further protein 4.1 binding. The demonstration of a specific binary association between protein 4.1 and actin suggests that this interaction contributes significantly to the stabilization of the spectrin-actin-protein-4.1 ternary complex (Morris, 1995).

Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered identify the sequences involved in spectrin-actin interactions, and to measure membrane strength. Gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Several SAB peptides appear to exhibit a weak, cooperative actin-binding activity that maps to the first 26 residues of the constitutive 59 aa. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlate with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without (in and of themselves) imparting unique function (Discher, 1995).

Protein 4.1 interaction with Glycophorin, a protein that shares a domain with Neurexin

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. Synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila Discs-large tumor suppressor protein (Marfatia, 1995)

The major attachment site for protein 4.1 on the human erythrocyte is glycophorin (GP) C/D. Purified protein 4.1 can bind to two distinct sites on glycophorin C/D. One of these interactions is direct, involving residues 82-98 on glycophorin C (61-77 on glycophorin D), while the other interaction is mediated by p55. The binding site for p55 on glycophorin C is localized to residues 112-128 (glycophorin D91-107). Another protein, known as Band 3, is bound by protein 4.1. The binding sites for band 3, glycophorin C/D, and p55 are all located within the 30-kDa domain of protein 4.1. The relative utilization of the three sites in normal membranes is 40% for p55, 40% for GPC/D, and 20% for band 3. The same region of protein 4.1 binds GPC/D and band 3, while the p55 binding site is distinct. The interactions involving protein 4.1 with p55 and p55 with GPC/D are of high affinity (nM), while those involving GPC/D and band 3 are 100-fold lower (microM). These results suggest that the most significant interactions between protein 4.1 and the membrane are those involving p55 (Hemming, 1995).

Protein 4.1 interaction with Band 3

In contrast to the detailed understanding of the role of protein 4.1 in regulating membrane mechanical properties through modulation of spectrin-actin interaction, very little is known regarding the functional implications of protein 4.1 interaction with band 3. Based on recent studies that have identified the sequence motif IRRRY in band 3 as the protein 4.1 interacting domain, the functional consequences of specific dissociation of band 3-protein 4.1 interaction by the synthetic peptide IRRRY were studied. Protein 4.1 bound to inside-out vesicles can be dissociated from band 3 but not from glycophorin C by IRRRY. Incorporation of IRRRY into resealed ghosts results in decreased membrane deformability and increased membrane mechanical stability. The observed alterations in membrane properties appears to result from increased band 3-ankyrin interaction following dissociation of protein 4.1 from band 3. These studies have enabled the identification of an important functional role for band 3-protein 4.1 interaction in modulating erythrocyte membrane properties (An, 1996).

A nuclear function for vertebrate protein 4.1

Nuclear matrices have been prepared from Madin-Darby canine kidney (MDCK) and HeLa cells. Two 4.1 polypeptides of 135,000 and 175,000 kDa may be associated with chromatin. Interestingly, nuclear matrices isolated after DNase I digestion contain a third protein 4.1 polypeptide (4.1p75), suggesting that it is a component of the nuclear matrix. The protein 4.1p75 (of molecular weight 75 kDa), is a common element of the nuclear matrix of mammalian cells. Moreover, 4.1p75 distributes to typical nuclear speckles that are enriched with the spliceosome assembly factor SC35. Protein 4.1p75 might be an anchoring element of the nucleoskeleton, playing a role similar to that described for the erythroid protein 4.1 in red blood cells (De Carcer, 1995).

Reconstructions of optical sections of human diploid fibroblast nuclei show 4.1 is intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes are detected in nuclear matrix. Several polypeptide bands can be demonstrated that are reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 is detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes are detected throughout the cell cycle but undergo dynamic spatial rearrangements during cell division. Protein 4.1 is observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function (Krauss, 1997).

To learn more about nuclear 4.1 protein, the nature of its association with the nuclear structure was analyzed in comparison with SC35 and snRNP antigens, splicing proteins of the nuclear speckle domains. When MDCK or HeLa cells are digested with DNase I and washed in the presence of high salt (2 M NaCl), snRNP antigens are extracted whereas protein 4.1 and SC35 remained colocalizing in nuclear speckles. In cells treated with RNase A or heat shocked, nuclear 4.1 distribution also resembles that of SC35. Experiments carried out in transcriptionally active nuclei shows that protein 4.1 distributes in irregularly shaped speckles that appeared to be interconnected. During transcriptional inhibition, protein 4.1 accumulates in rounded speckles lacking interconnections. When cells are released from transcriptional inhibition, protein 4.1 redistributes back to the interconnected speckle pattern of transcriptionally active cells, as it was also observed for SC35. Coprecipitation of 4.1 and SC35 proteins from RNase A digested HeLa nuclei further indicates that these two proteins are associated, forming part of the nuclear speckle domains to which they attach more tightly than snRNP antigens (Lallena, 1997).