Filamins: promiscuous organizers of the cytoskeleton

Filamins are elongated homodimeric proteins that crosslink F-actin. Each monomer chain of filamin comprises an actin-binding domain, and a rod segment consisting of six (Dictyostelium filamin) up to 24 (human filamin) highly homologous repeats of approximately 96 amino acid residues, which adopt an immunoglobulin-like fold. Two hinges in the rod segment, together with the reversible unfolding of single repeats, might be the structural basis for the intrinsic flexibility of the actin networks generated by filamins. There are numerous filamin-binding proteins that associate, in most cases, along the repeats of the rod repeats. This rather promiscuous behaviour renders filamin a versatile scaffold between the actin network and finely tuned molecular cascades from the membrane to the cytoskeleton.     

Comparison of mRNA and protein levels of four members of the protein 4.1 family: the type II brain 4.1/4.1B/KIAA0987 is the most predominant member of the protein 4.1 family in rat brain

The human gene for the fourth member of the protein 4.1 family, KIAA0987, was recently identified by comprehensive cDNA analysis. To further characterize the corresponding gene and its product in rats, we cloned and sequenced rat KIAA0987 cDNA. RNA blot analyses revealed that the rat KIAA0987 gene was abundantly expressed only in the brain, kidney, and testis. Although we have previously reported that the third member of the protein 4.1 family, the KIAA0338 gene product, is predominantly expressed in rat brain, and thus was named brain 4.1, quantitative RNA blot analyses indicated that KIAA0987 should be called something other than brain 4.1 because the level of KIAA0987 mRNA was found to be of the same order as that of KIAA0338 mRNA. Our quantitative immunoblot analysis showed that the most predominant member of the protein 4.1 family at the protein level was the product of the KIAA0987 gene, not that of the KIAA0338 gene. Taking these results together, we consider it reasonable to name the KIAA0338 and KIAA0987 gene products 'type I brain 4.1' and 'type II brain 4.1,' respectively, because these two products were found to be more prominently produced in rat brain than the other two members of the protein 4.1 family, erythroid 4.1 and 4.1G.     

The WASP-WAVE protein network: connecting the membrane to the cytoskeleton

Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins are scaffolds that link upstream signals to the activation of the ARP2/3 complex, leading to a burst of actin polymerization. ARP2/3-complex-mediated actin polymerization is crucial for the reorganization of the actin cytoskeleton at the cell cortex for processes such as cell movement, vesicular trafficking and pathogen infection. Large families of membrane-binding proteins were recently found to interact with WASP and WAVE family proteins, therefore providing a new layer of membrane-dependent regulation of actin polymerization.     

The Protein 4.1 family: Hub proteins in animals for organizing membrane proteins

Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and differential mRNA splicing. Finally, the spectrum of interactions of the 4.1 proteins overlaps with that of another membrane-cytoskeleton linker, ankyrin. Both ankyrin and 4.1 link to the actin cytoskeleton via spectrin, and we hypothesize that differential regulation of 4.1 proteins and ankyrins allows highly selective control of cell surface protein accumulation and, hence, function. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé     

The secretory carrier membrane protein family: structure and membrane topology

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.     

The synthesis and accumulation of membrane protein 4.1 in Friend erythroleukemia cells

The effect of extensive differentiation on the synthesis and accumulation of protein 4.1 were studied on Friend erythroleukemia cells grown in suspension and on fibronectin coated dishes. Whole membranes of Friend erythroleukemia cells (FELC) contained a protein 4.1a and 4.1b doublet of Mr 76 and 74 kDa and two minor bands of Mr 105 and 43 kDa that cross-reacted with anti-human protein 4.1 IgG. These proteins were present even in uninduced cells. The synthesis of protein 4.1 was maximal after 4 days of induction in both suspension culture and in fibronectin-coated dishes whereas the protein 4.1 continued to accumulate until the seventh day. More protein 4.1 accumulated in cells grown on fibronectin-coated dishes, at each stage of differentiation, than in cells grown in suspension. The protein 4.1a/4.1b ratio changed during differentiation. The amounts of protein 4.1b increased progressively after induction until the protein 4.1a/4.1b ratio was similar to that of mouse mature erythrocyte. The protein 4.1a/4.1b ratio appears to be an internal marker of erythroid differentiation.     

Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane

Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system.     

Nectin-like molecule 1 is a protein 4.1N associated protein and recruits protein 4.1N from cytoplasm to the plasma membrane

Nectins are immunoglobulin superfamily adhesion molecules that participate in the organization of epithelial and endothelial junctions. Sharing high homology with the poliovirus receptor (PVR/CD155), nectins were also named poliovirus receptor-related proteins (PRRs). Four nectins and five nectin-like molecules have been identified. Here we describe the cloning and characterization of human and mouse nectin-like molecular 1 (NECL1). Human and mouse NECL1 share 87.3% identity at the amino acid level. NECL1 contains an ectodomain made of three immunoglobulin-like domains, and a cytoplasmic region homologous to those of glycophorin C and contactin-associated protein. RNA blot and in situ hybridization analysis showed that NECL1 predominantly expressed in the central nervous system, mainly in neuronal cell bodies in a variety of brain regions including the cerebellum, cerebral cortex and hippocampus. In vitro binding assay proved the association of NECL1 with protein 4.1N. NECL1 localizes to the cell-cell junctions and recruits protein 4.1N to the plasma membranes through its C-terminus, thus may regulate the function of the cell-cell junction. We propose that the NECL1 and protein 4.1N complex is involved in the morphological development, stability, and dynamic plasticity of the nervous system.     

The postsynaptic spectrin/4.1 membrane protein "accumulation machine"

An important aspect of the function of the membrane-associated cytoskeleton has been suggested to be to trap and retain selected transmembrane proteins at points on the cell surface specified by cell adhesion molecules. In the process, cell adhesion molecules are cross-linked to each other, and so junctional complexes are strengthened. In this short review, we will discuss recent advances in understanding the role of this "accumulation machine" in postsynaptic structures. Function in the brain depends on correct ordering of synaptic intercellular junctions, and in particular the recruitment of receptors and other apparatus of the signalling system to postsynaptic membranes. Spectrin has long been known to be a component of postsynaptic densities, and recent advances in molecular cloning indicate that beta spectrins at PSDs are all "long" C-terminal isoforms characterised by pleckstrin homology domains. Isoforms of protein 4.1 are also present at synapses. All four 4.1 proteins are represented in PSD preparations, but it is 4.1R that is most enriched in PSDs. 4.1R binds to several proteins enriched in PSDs, including the characteristic PSD intermediate filament, alpha-internexin. Both 4.1 and spectrin interact with ionotropic glutamate receptors (AMPA and NMDA receptors, respectively): 4.1 stabilises AMPA receptors on the cell surface. By linking these receptors to the cytoskeletal and cell adhesion molecules that specify glutamatergic synapses, the membrane protein accumulation machine is suggested to direct the formation of postsynaptic signalling complexes.     

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

Background

Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes.

Case presentation

All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced.

Conclusion

A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission.     

Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1- like protein

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin- binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.     

Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.     

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.     

Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other hand, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The C1q complement family of synaptic organizers: not just complementary

1. Download : Download high-res image (198KB)
2. Download : Download full-size image

     

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily

The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.     

The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.