Protein 4.1-mediated membrane targeting of human discs large in epithelial cells

Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R.     

Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55.

Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA.     

cDNA cloning, genomic organization and expression of the novel human metallophosphoesterase gene MPPE1 on chromosome 18p11.2

We have isolated a 1,926-bp cDNA that encodes a novel polypeptide of 396 amino acid residues with a calculated molecular mass of 45.2 kDa. This MPPE1 polypeptide consists of a predicted signal sequence of 45 residues at the N-terminus, a 240-amino acid metallo-phosphoesterase domain, and a 24-amino acid transmembrane domain at the C-terminus. The genomic organization of the human MPPE1 gene proved to consist of 14 exons and to span about 27 kb. The gene was located on chromosome 18p11.2, adjacent to the G protein Golf alpha gene (GNAL), in tail-to-tail orientation, partially overlapping with the 3' UTR of the latter gene. MPPE1 is expressed as an mRNA of 2.2 kb in the brain, but not in any other tissues studied here. 3' RACE analysis defined a single functional polyadenylation site within the 3' UTR of the GNAL gene, while RT-PCR analysis revealed an alternatively spliced form of MPPE1, which included an additional exon located within the last intron. The alternatively spliced form encoded a truncated variant of MPPE1 with a calculated molecular mass of 38.8 kDa that lacks the C-terminal transmembrane domain.     

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.     

Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane

Three binary protein-protein interactions, glycophorin C (GPC)-4.1R, GPC-p55, and p55-4.1R, constitute the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little is known regarding the molecular basis for the interaction of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R constitute the binding interfaces for GPC and p55, respectively. We further show that 4.1R increases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaction between p55 and GPC. Finally, we document that binding of calmodulin to 4.1R decreases the affinity of 4.1R interactions with both p55 and GPC in a Ca(2+)-dependent manner, implying that the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation in the erythrocyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary complex in the erythrocyte membrane.     

Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.     

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.     

Cyclin H is a new binding partner for protein kinase CK2

PTP-FERM is a protein tyrosine phosphatase (PTP) of Caenorhabditis elegans containing a FERM domain and a PDZ domain. Here we report the characterization of PTP-FERM and the essential role of its FERM domain in the localization of PTP-FERM in the worm. There are at least three alternatively spliced PTP-FERM isoforms, all of which contain a band 4.1/FERM domain, a PDZ domain, and a catalytic domain. PTP-FERM possessed phosphatase activity. PTP-FERM was expressed predominantly in neurons in the nerve ring and the ventral nerve cord. PTP-FERM was found in the nerve processes and to be enriched in the peri-membrane region. Studies using various deletion mutants revealed that the FERM domain was essential and sufficient for the subcellular localization. These results suggest the essential role of the FERM domain in the function of PTP-FERM in the neurons of C. elegans.     

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 multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.     

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.     

Genomic organization and alternative promoter usage of the two thyroid hormone receptor beta genes in Xenopus laevis.

Each of the two Xenopus laevis thyroid hormone receptor beta genes is at least 70 kilobases in length with similar intron-exon organization. There are up to eight alternatively spliced exons in the 5'-untranslated region. Excluding the extreme amino terminus, each receptor is encoded by six exons spanning about 6 kilobases of the genome, in which each of the two zinc fingers that comprise the DNA-binding domain is encoded by a separate exon and the hormone-binding domain is split into three exons. The last exon of the coding region also contains at least 600 base pairs of the 3'-untranslated region, which is about 8 kilobases. Each of the receptor genes has two promoters and just one of them is up-regulated in tadpoles by the administration of thyroid hormone.     

Mutational analysis of cell cycle inhibition by integrin beta1C

Integrin beta1C is an alternatively spliced cytoplasmic variant of the beta1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by beta1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of beta1C (residues 732-805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the beta1A, beta1B, beta1D, beta3, and beta5 cytoplasmic domains had weak and variable effects. The beta1C cytoplasmic domain is composed of a membrane proximal region (732-757) common to all beta1 variants and a COOH-terminal 48-amino acid domain (758-805) unique to beta1C. The beta1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by beta1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the beta1C-specific domain showed that the 18-amino acid sequence 775-792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally, beta1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/beta1C (758-805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for beta1C as a growth inhibitor in vivo.