Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Ig domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumor suppressor candidate gene commonly deleted in certain human cancer tissues.     

Fibronectin type III repeats mediate RGD-independent adhesion and signaling through activated beta1 integrins

Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of beta1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and beta1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated beta1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.     

Structural features of a close homologue of L1 (CHL1) in the mouse: a new member of the L1 family of neural recognition molecules

We have identified a close homologue of L1 (CHL1) in the mouse. CHL1 comprises an N-terminal signal sequence, six immunoglobulin (Ig)-like domains, 4.5 fibronectin type III (FN)-like repeats, a transmembrane domain and a C-terminal, most likely intracellular domain of approximately 100 amino acids. CHL1 is most similar in its extracellular domain to chicken Ng-CAM (approximately 40% amino acid identity), followed by mouse L1, chicken neurofascin, chicken Nr-CAM, Drosophila neuroglian and zebrafish L1.1 (37-28% amino acid identity), and mouse F3, rat TAG-1 and rat BIG-1 (approximately 27% amino acid identity). The similarity with other members of the Ig superfamily [e.g. neural cell adhesion molecule (N-CAM), DCC, HLAR, rse] is 16-11%. The intracellular domain is most similar to mouse and chicken Nr-CAM, mouse and rat neurofascin (approximately 60% amino acid identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian and zebrafish L1.1 and L1.2 (approximately 40% amino acid identity). Besides the high overall homology and conserved modular structure among previously recognized members of the L1 family (mouse/human L1/rat NILE; chicken Ng-CAM; chicken/mouse Nr-CAM; Drosophila neuroglian; zebrafish L1.1 and L1.2; chicken/mouse neurofascin/rat ankyrin-binding glycoprotein), criteria characteristic of L1 were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig-like domains and FN-like repeats. These show a collinearity in the six Ig-like domains and four adjacent FN-like repeats that is remarkably conserved between L1 and molecules containing these modules (designated the L1 family cassette), including the GPI-linked forms of the F3 subgroup (mouse F3/chicken F11/human CNTN1; rat BIG-1/mouse PANG; rat TAG-1/mouse TAX-1/chicken axonin-1). The colorectal cancer molecule (DCC), previously introduced as an N-CAM-like molecule, conforms to the L1 family cassette. Other structural features of CHL 1 shared between members of the L1 family are a high degree of N-glycosidically linked carbohydrates (approximately 20% of its molecular mass), which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kDa band and smaller fragments of 165 and 125 kDa. As for the other L1 family members, predominant expression of CHL1 is observed in the nervous system and at later developmental stages. In the central nervous system CHL1 is expressed by neurons, but, in contrast to L1, also by glial cells. Our findings suggest a common ancestral L1-like molecule which evolved via gene duplication to generate a diversity of structurally and functionally distinct yet similar molecules.     

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.     

The evolution of titin and related giant muscle proteins

Titin and twitchin are giant proteins expressed in muscle. They are mainly composed of domains belonging to the fibronectin class III and immunoglobulin c2 families, repeated many times. In addition, both proteins have a protein kinase domain near the C-terminus. This paper explores the evolution of these and related muscle proteins in an attempt to determine the order of events that gave rise to the different repeat patterns and the order of appearance of the proteins. Despite their great similarity at the level of sequence organization, titin and twitchin diverged from each other at least as early as the divergence between vertebrates and nematodes. Most of the repeating units in titin and twitchin were estimated to derive from three original domains. Chicken smooth-muscle myosin light-chain kinase (smMLCK) also has a kinase domain, several immunoglobulin domains, and a fibronectin domain. From a comparison of the kinase domains, titin is predicted to have appeared first during the evolution of the family, followed by twitchin and with the vertebrate MLCKs last to appear. The so-called C-protein from chicken is also a member of this family but has no kinase domain. Its origin remains unclear but it most probably pre-dates the titin/twitchin duplication.     

Mechanism of nicotinic acetylcholine receptor cluster formation by rapsyn

Rapsyn, a peripheral membrane protein of skeletal muscle, clusters nicotinic acetylcholine receptors (nAChRs) at high density in the postsynaptic membrane. The mechanism of nAChR clustering by rapsyn was analyzed by expressing nAChRs in HEK293T cells with various fragments of mouse rapsyn fused to green fluorescent protein. Membrane targeting of rapsyn is conferred solely by its acylated N terminus, as the myristoylated N-terminal 15 amino acids of rapsyn are sufficient to target green fluorescent protein to the plasma membrane. However, neither N-terminal myristoylation nor the conserved N-terminal amino acid sequence is essential. Membrane targeting, self-association, and nAChR clustering are preserved when the first 10 amino acids of rapsyn were replaced by those of src, which also contains a consensus sequence for N-myristoylation, or by those of GAP43, which contains a palmitoylation sequence. Rapsyn1-90, containing two tetratrichopeptide repeats is sufficient for self-association. Rapsyn1-360, lacking the cysteine rich domain, clusters nAChRs, while rapsyn1-287, containing seven tetratrichopeptide repeats, does not cluster nAChRs. We identified rapsyn298-331 as a potential coiled-coil domain, and established that mutations disrupting coiled-coil propensity prevent nAChR clustering. Thus the structural domains of rapsyn necessary for membrane targeting, self-association, and nAChR clustering are distinct, with nAChR-rapsyn interaction mediated by a previously unrecognized coiled-coil motif.     

Facial nerve paralysis after intratemporal and extratemporal blunt trauma

Tenascins (TNs) are a family of extracellular matrix glycoproteins. The first member of this family to be recognized, tenascin-C (TN-C), is known to be expressed in various tumors including human astrocytomas. Tenascin-X (TN-X) is the latest member of the TN family to be reported, and its expression in tumor tissues has not yet been examined. In this study, we found expression of TN-X in glioma cell lines and human astrocytomas by immunoblot analysis using anti-mouse TN-X antibodies. We also examined the expression of TN-C and TN-X immunohistochemically in a series of 32 human astrocytomas and tissue from 5 normal brains. Expression of TN-X was up-regulated to a higher degree in low-grade astrocytomas than in high-grade astrocytomas. TN-X was mainly localized in the perivascular stroma around tumor vessels, and weakly expressed in the intercellular spaces among tumor cells. In contrast, TN-C was more strongly expressed in the intercellular spaces and in tumor vessels in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas) than in low-grade astrocytomas. In the tissues expressing both TNs, the distribution of TN-X was often reciprocal to that of TN-C. These findings indicate that the expression of TN-C and TN-X in astrocytomas is different, and that these glycoproteins could be involved in neovascularization in different manners.     

The light chain-binding domain of the smooth muscle myosin heavy chain is not the only determinant of regulation

Interactions between the dephosphorylated regulatory light chains (RLCs) of smooth muscle myosin are involved in maintaining the enzymatically "off" state. Expressed chimeric smooth muscle heavy meromyosins containing skeletal muscle myosin heavy chain (HC) sequences were used to assess the relative importance of the light chain-binding domain (or "neck") to regulation. Surprisingly, regulation remained intact with a skeletal RLC-binding site. A chimera with the entire alpha-helical neck composed of skeletal HC sequence showed 2-fold regulation of motility and nearly 5-fold regulation of actin-activated ATPase activity. Complete activation of the dephosphorylated state (i.e. complete loss of regulation) occurred when skeletal HC sequence extended from the head/rod junction to the SH1-SH2 helix. Smooth muscle-specific sequences near the motor domain may therefore position the regulatory domain in a way that optimizes RLC-rod-head interactions, thus enabling a completely off state when the RLC is dephosphorylated. Conversely, a chimera that joins the motor domain from unconventional myosin V to the smooth muscle myosin neck and rod showed only 2-fold regulation. The presence of the smooth muscle light chain-binding region and rod is therefore not sufficient to confer complete phosphorylation-dependent regulation upon all motor domains of the myosin family.     

Identification and expression of two novel CLIP-170/Restin isoforms expressed predominantly in muscle

CLIP-170 and Restin, microtubule-binding proteins originally cloned from human cells, are identical except for a stretch of 35 amino acids present in Restin, but missing from CLIP-170. Here we present the discovery of two novel isoforms of the CLIP-170/Restin gene in both chickens and humans. One of the new isoforms, named CLIP-170(11), contains an 11 amino acid insert instead of the 35 amino acid insert found in Restin. Eight of these 11 amino acids, including a helix-breaking proline residue, are perfectly conserved between chickens and humans. The second new isoform, named CLIP-170(11+35), contains both the 11 and 35 amino acid inserts in tandem. PCR analysis of chicken genomic DNA revealed that all four isoforms result from differential splicing of two exons in a region of the CLIP-170 gene that contains approximately 8.6 kb of intervening sequence. We found that the CLIP-170(11) and CLIP-170(11+35) are expressed preferentially in muscle tissues. Chicken and human skeletal muscle express predominantly CLIP-170(11) and to a lesser extent CLIP-170 and CLIP-170(11+35). Adult chicken cardiac and smooth muscles also express CLIP-170(11) and CLIP-170(11+35), but CLIP-170 is the predominant isoform in these muscles as it is in all other tissues except brain. The ratios of CLIP-170 isoform expression found in embryonic and adult chicken cardiac muscles reveal that isoform expression is regulated differentially in different developmental stages as well as in different tissues.     

Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system

The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of alpha 7B in the myocardium. In addition to striated muscle, alpha 7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.     

The fibrinogen-like globe of tenascin-C mediates its interactions with neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta

Two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan (the extracellular domain of protein-tyrosine phosphatase-zeta/beta), are high-affinity ligands of tenascin-C. Using portions of tenascin-C expressed as recombinant proteins in human fibrosarcoma cells, we have demonstrated both by direct radioligand binding assays and inhibition studies that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen-like globe and that phosphacan binds to this single domain with nearly the same affinity (Kd approximately 12 nM) as to native or recombinant tenascin-C. However, maximum binding of neurocan requires both the fibrinogen globe and some of the adjacent fibronectin type III repeats. Binding of phosphacan and neurocan to intact tenascin-C, and of phosphacan to the fibrinogen globe, is significantly increased in the presence of calcium. Chondroitinase treatment of the proteoglycans did not affect their binding to either native tenascin-C or to any of the recombinant proteins, demonstrating that these interactions are mediated by the proteoglycan core proteins rather than through the glycosaminoglycan chains. These results are also consistent with rotary shadowing electron micrographs that show phosphacan as a rod terminated at one end by a globular domain that is frequently seen apposed to the fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma cells adhere to and spread on deletion variants of tenascin-C containing only the epidermal growth factor-like domains or the fibronectin type III repeats and the fibrinogen globe. In both cases cell adhesion was inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C.     

Molecular cloning of the cDNA encoding human skeletal muscle triadin and its localisation to chromosome 6q22-6q23

We have cloned and sequenced the cDNA encoding triadin, a junctional terminal cisternae protein from human skeletal muscle. The cDNA, 2941 base pairs in length, encodes a protein of 729 amino acids with a predicted molecular mass of 81,545 Da. Hydropathy analysis indicates that triadin of human skeletal muscle has the same topology in the myoplasmic, transmembrane and sarcoplasmic reticulum luminal domains as that of triadin from rabbit skeletal muscle. The number and relative position of potential modulation sites are also conserved between the human and rabbit proteins. The cDNA sequence of the predicted sarcoplasmic reticulum luminal domain of human triadin diverged from that of rabbit, with an observed similarity of 82%, translating to an identity of 77% in amino acid sequence. Two insertions of 9 and 12 residues in the amino acid sequence were observed in the predicted luminal domain of triadin, although the structural and functional consequences of such insertions are expected to be minimal. Using fluorescence in situ hybridisation, we have assigned the gene encoding human triadin to the long arm of chromosome 6 in the region 6q22-6q23. Our structural analysis of human triadin supports a central role for this protein in the mechanism of skeletal muscle excitation/contraction coupling.     

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.     

Molecular cloning and characterization of human non-smooth muscle calponin

cDNA clones encoding a calponin isoform with 309 amino acids have been isolated from human heart. The deduced amino acid polypeptide (M(r) 33,697) showed a neutral isoelectric point of 7.1. The mRNA, expressed in cultured smooth muscle cells as well as in fibroblasts, vascular endothelial cells, and keratinocytes, contains a 3' untranslated region of 1.2 kilobases that includes an Alu repetitive sequence in the antisense direction. On the basis of the nucleotide sequence identity to an expressed sequence tag, HUM21ES93 [Cheng, J.-F., Boyartchuk, V., and Zhu, Y. (1994) Genomics 23, 75-84], the human neutral calponin gene is assigned to chromosome 21q11.1. The amino acid sequence indicates that this protein is the human equivalent of mouse calponin-h2 (94.8% identity) [Strasser, P., Gimona, M., Moessler, H., Herzog, M., and Small, J.V. (1993) FEBS Lett. 330, 13-18]. Three tandem repeats of 29 amino acids, a Vav-homologous region and an actin-binding sequence, originally identified in the basic calponin isoform, are conserved. There are two consensus phosphorylation sites for tyrosine kinase. An immunoreactive form of the neutral calponin appears to be localized with vinculin in the cell-to-cell junctions of cardiomyocytes. Mouse calponin-h2 is also expressed in both embryonic and adult heart. These results indicate that the human neutral calponin is a non-smooth muscle isoform, and may play a physiological role in cytoskeletal organization.