Forss-Petter, S., Danielson, P. & Sutcliffe, J. G. (1 986) J. Neurosci. Fort, P.. Rech, J., Vie, A., ... latter category. cell adhesion molecules can be defined as calcium ...
625th MEETING, LONDON Amersham International p.1.c. ( 1987) MI3 Cloning and Sequencing Handbook Bishop, M. & Thompson, E. (1984) Nucleic Acids Res. 12, 547 1-5474 Clemens. M. J. ( 1 987) Cell 49, 157-1 58 Creighton, T. E. ( 1984) Methods Enzymol. 107,305-329 Davies, K. E. (ed.) ( 1 986) Hicman Genetic Diseases: A I’rui,tical Approach, IRL Press, Oxford Day, 1. N. M. ( I987a) Ph.D. Thesis, Cambridge University, U.K. Day, 1. N. M. ( 19876)Mol. Cell Probes 1,275-295 Day, I. N. M. & Thompson, R. J. (1984) Biochem. Soc. Trans. 14, 350-35 1 Day, 1. N. M. &Thompson, R. J. (1987) FEBS Lett. 210, 157-160 Day, 1. N. M., Allsopp, M. T. E. P., Moore, D. C. McN. & Thompson, R. J.(1987a) FEBSLert. 222, 139-143 Day, I. N. M., Allsopp, M. T. E. P., Moore, D. C. McN. & Thompson, R. J. (1988) Biochem. Soc. Trans. 16, in the press Dayhoff. M. 0.. Barker, W. C. & Hunt, L. T. (1983) Methods Enzymol. 91,524-545 Doran, J. F., Jackson, P., Kynoch, P. A. M. & Thompson, R. J. ( 1983)J. Neurochem. 40, 1542- 1547 Forss-Petter, S., Danielson, P. & Sutcliffe, J. G. ( 1 986) J. Neurosci. Res. 16.141-156 Fort, P.. Rech, J., Vie, A., Diechaczyk, M.. Bonnieu, A., Jeanteur, P. & Blanchard, J-N. (1987)Nucleic Acids Res. 15,5657-5667 Garnier, J., Osgusthorpe, D. J. & Robson, B. (1978) J. Mol. Biol. 120.97- 120 Giallongo, A.. Feo, S., Moore, S., Croce, C. M. & Showe, L. (1986) Proc. Null. Acad. Sci. U.S.A. 83, 6741-6745 Hooper, C. M., Allsopp, M. T. E. P. & Day, 1. N. M. ( 1988) Biochem. Soc. Trans. 16. in the press Isselbacher. K. J., Adams, R. D.. Braunwald, E., Petersdorf, R. G. & Wilson, J. D. ( 1980) Harrisons Principles of Internal Medicine, 9th edn., McGraw-Hill Kogakusha Ltd., Tokyo
457 Jackson, P. & Thompson, R. J. ( 1 98 1 ) J. Neurol. Sci. 49,429-438 Le Douarin, N. M. (1982) The Neural Crest, Cambridge University Press, Cambridge, U.K. Maniatis, T., Fritsch, E. F. & Sambrook, J. ( 1982) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Publications, New York Marangos, P. J. & Schmechel, D. ( 1 987) Annu. Rev. Neurosci. 10, 269-295 Marangos, P. J., Parma, A. M. & Goodwin, F. K. (1978)J. Neurochem. 31,727-732 Milner, R. J. & Sutcliffe, J. G. (1983) Nucleic Acids Res. 5, 1729- 1739 Morgan, J. I. & Curran, T. ( 1 986) Nature (London) 322,552-555 Nathans, J., Thomas, D. & Hogress, D. S. (1986) Science 232, 192-202 Pearse, A. G. E. (1980) Clin. Endocrinol. Metab. 9 , 2 1 1-222 Rogers, J. H. (1983) Nature (London) 301,460 Sakimura, K., Kushiya, E., Obinata, M. & Takahashi, Y. ( 1 985) Nucleic Acids Res. 13,4365-4378 Schlesinger, M. J. (1986)J. Cell Biol. 103,32 1-325 Schmechel, D., Marangos, P. J. & Brightman, M. (1978) Nature (London) 276,834-836 Shaw, G. & Kamen, R. (1986) Cell 46,659-667 Staden, R. ( 1 982) Nucleic Acids Res. 10,295 1-296 1 Thompson, R. J. & Day, 1. N. M. (1988) in Neurobiological Research, vol. I1 (Marangos, P. J., Campbell, 1. & Cohen, R. M., eds.), pp. 209-228, Academic Press, California Thompson, R. J., Doran, J. F., Jackson, P., Dhillon, A. P. & Rode, J. (1983) Brain Res. 278,224-228 Treisman, R. (1985) Cell 42,889-902
Received 8 December 1987
Structure and expression of neural cell adhesion molecule complementary DNA clones in skeletal muscle C. HOWARD BARTON, GEORGE DICKSON: HILARY J. G O W E R and F R A N K S. WALSH Departmetit o f Neirrochernistvy, Iristitrite of Neurology, Queen Square, London WCIN 3RG, U . K . Tissue morphogenesis results from the formation, migration and differentiation of distinct cell lineages in precise spatiotemporal patterns. Differential cell-cell and cell-substratum interactions are thought to play a crucial role in these developmental events, and indeed, at the biochemical level, a diverse array of cell-surface and extracellular-matrix ( E C M ) antigens mediating cellular adhesion and recognition events have been described (see Garrod, 1986 for review). In recent years, however, molecular genetic analyses have allowed broad groupings o f similar adhesion molecules, homologous at functional and/or structural levels, to be defined. Thus, there arc specific Components of E C M such as fibronectin and laminin, cell-surface receptors for these components such as the integrin family (Hynes, 1987), and surface components involved in direct cell-cell bond formation. In this latter category. cell adhesion molecules can be defined as calcium dependent such as the cadherin family (Takeichi, 1987). o r calcium independent and belonging to the Ig gene superfamily, e.g. neural cell adhesion molecule (N-CAM) (Cunningham et a/., 19X7), myelin-associated glycoprotein (MAG) (Salzer, 1987) lymphocyte-function-associated antigen (LFA-3) (Seed, 1987) or other structurally unrelated Abbreviations used: ECM. extracellular matrix; N-CAM, neural cell adhesion molecule; MAG, myelin-associated glycoprotein.
families such as Ng-CAM. Table 1 is a list of the better characterized families of cell adhesion molecules and attempts to categorize them o n the basis of similarity in structure or function. T h e group of related glycoproteins known collectively as N-CAM have been widely studied and are thought to play a role in neurogenesis, axon guidance and the formation and innervation of skeletal muscle (Rutishauser et ul., 1978; Fraser et a[., 1984; Rutishauser et al., 1983). While modulation of N-CAM structure at the levels of R N A processing and post-translational modification have been described, correlations with the diverse functional roles of this group of molecules remain, as yet, undefined. To identify structural interrelations between N-CAM isoforms expressed in skeletal muscle, and as a prelude to direct analysis of structure-function relationships via eukaryotic gene transfer and site-directed mutagenesis studies, we have isolated and characterized complementary D N A s spanning the coding regions for several human muscle N-CAM cDNAs. A c D N A library was generated in I g t 1 1 from human fetal muscle poly(A+) mRNA and was screened by filter plaque hybridization with two c D N A probes called pM 1.3 and 9 1 1 (both generous gifts from Dr C. Goridis). Probe 911 was initially isolated from a mouse brain c D N A library using an oligonucleotide probe to the derived N-terminal protein sequence (C. Goridis, personal communication). This probe contains both coding and 5‘ untranslated sequence, whereas the 3‘ probe pM1.3 (Goridis et al., 1985) was originally isolated from a similar c D N A library by immunoscreening with a mouse brain monoclonal antibody.
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onstrated for other proteins (Boothroyd et al., 1981; Weiss et al., 1986), which are attached to membrane by phosphaList of cell adhesion molecule families. Cell adhesion molecules tidylinositol. N-CAM- 125 and N-CAM- 155 isoforms have can be divided into two main groups, namely cell adhesion been previously shown to be released from mouse G 8 molecules and substrate adhesion molecules. A major problem myotubes in culture by phospholipase C (Moore et af., in surveying the literature on adhesion molecules is that there is 1987);therefore, it is likely that this full-length clone encodes no agreed nomenclature for these molecules and molecules of a human analogue of a non-transmembrane, phosphatidylsimilar structure are called different names in diffrent labora- inositol-linked N-CAM isoform. The central region of the tories. Further details of the molecules mentioned in the list can sequence contains six consensus sites for N-linked glycosylabe found in Thiery & Edelman (1985), Hynes (1987) and tion. The N-terminal region of the molecule contains 10 Williams (1987). cysteines residues about 6 0 residues apart. These 10 cysteines can be subdivided into five groups which exhibit signiA. Cell adhesion molecule (CAMS) ficant homology with each other. N-CAM has no free SH (i) Ca2+-dependent adhesion (requires Ca2+ for action and groups (Hoffman et al., 1982) and since none of them has Ca2 gives protease resistant property) been shown to be involved in interchain disulphide bridges, L-CAM (E-Cadherin; Uvomorulin; Cell CAM120/ it is likely that the cysteine residues are implicated in five 80; ARC- 1; rr- 1 antigen) intramolecular or more probably intradomain bridges. This folding pattern of repetitive intramolecular disulphide N-Cadherin (A-CAM;N-CAL-CAM) bridges is common to members of the immunoglobulin gene (ii) Ca2+-independentadhesion superfamily (Williams, 1987). The alignment of these five ( a ) Immunoglobulin superfamily members N-CAM domains around the centrally placed cysteine N-CAM; MAG; Po; CEA; (structuralsimilarity) residues reveals five additional invariant residues, four PDGFR; CSFIR residues maintained in four domains and another 13 con(6) Ng-CAM (NILE, L l ) ; J1; cell CAM 105 ( n o implied served in only three domains. The N-CAM immunoglobulin structural similarity) domains have characteristics of constant Ig homology units in the size of the domains (100 residues) and the spacing of B. Substrateadhesion molecules the cysteines. However, the N-CAM sequences in all five ( i ) Extracellular matrix components, e.g. fibronectin; laminin; domains resemble those of the variable Ig homology unit, heparin sulphate proteoglycan, collagen (no implied struc- particularly around the second cysteine. A new grouping, the tural similarity) C2 set, accommodating such domain structures has recently (ii) ECM-receptor family that recognizes RDGS, e.g. CSAT; been proposed (Williams, 1987) and includes members such LFA-1; MAC- 1 (structuralsimilarity) as C E A (Beauchemin et af., 1987), MAG (Salzer et al., 1987) as well as N-CAM. These cell surface glycoproteins exhibit multidomain structures like N-CAM and alignment of the domains reveals a high degree of similarity clustered around A number of clones were isolated, most of which were the pairs of cysteine residues. Analysis of N-CAM genomic unique to each probe. However, one clone was isolated with clones from the chick (Owens et af., 1987) has shown that the 5’ probe, 1 1 , which contained one large EcoRl fragment each of these domains is encoded by two exons, whereas for of 1.7 kb and one smaller EcoRl fragment of 1.2 kb. The immunoglobulin each domain is derived from a discreet smaller EcoRl fragment was common to both pools. These exon. It is conceivable that N-CAM and the other proteins two EcoRl fragments were subcloned into M13mp18 possessing the C2 domain structure evolved from an immu(Messing, 1983) in both orientations. Following Xbal/Sphl noglobulin precursor gene before the divergence of the double digestion of these plasmids, serial deletions were con- immunoglobulin C and V region domains. An additional extracellular coding domain (MSD 1 ) was structed in both directions by using exonuclease 111 and S1 nuclease (Henikoff, 1984). These subclones were then observed towards the 3’ end of the full-length human muscle sequenced by dideoxy chain-termination methodology cDNA clone. This sequence block encodes 37 amino acids (Sanger et al., 1977). Individual gel tracks were aligned and with 7 proline, 6 serine and 5 threonine and MSDl is not whole sequences were compared for similarity with other present in any chick or mouse cDNA isolated to date from sequences in the NBRL database. Orientation of the cDNA brain (Dickson et af., 1987). Another human muscle cDNA subclones was ascertained by Northern blot hybridization clone of 1.6 kb, 14.4, has been isolated and sequenced in which the block of 37 amino acids of MSDl is replaced by with single-stranded M13 probes. The entire 2799 bp cDNA sequence of this clone was an arginine codon. Comparison of chick genomic sequence highly homologous (76%) with the published cDNA indicates that the MSDl is inserted at a recognized splice sequence of a mouse brain 2.9 kb transcript encoding a non- junction (Owens et af., 1987). The MSDl sequence is the transmembrane NCAM-120 isoform (Barthels et af., 1987) first major variation in primary sequence to be reported in and the percentage homology increased to 88% at the amino the extracellular coding sequence of any N-CAM isoform, acid level. The major open reading frame of 1 1 encoded a and is contrary to the view that NCAM diversity is only 761 amino acid protein of 83 kd (Fig. 1)and left 125 bases achieved by alternative mRNA splicing within the intraceland 3 19 bases of 5’ and 3’ untranslated sequence, respect- M a r domain or at the plasma membrane (Hemperly et af., ively. The first ATG codon was preceded by two of the five 1986). Further comparison of the 3’ EcoRl fragment of 11 with a base initiation consensus sequences (Kozak, 1984). The predicted signal peptide of 19 predominantly hydrophobic clone called 14.4 indicates an additional point of divergence amino acids was identical to that of the mouse brain non- 630 bp from the 5‘ end of this clone. While the reading frame transmembrane isoform, except for a substitution of of 11 leads to a hydrophobic region and a stop codon, 14.4 arginine by glutamine at position 3. The first amino acid of encodes an additional 146 amino acids. Within this region the mature protein after signal cleavage is leucine in common there is a 19 amino acid hydrophobic membrane spanning with that of the mouse brain isoform. Hydropathy analysis region and an intracellular domain of 118 amino acids. revealed, in addition to the signal peptide, another region of Clone 14.4 thus encodes the C-terminal region of a human 20 hydrophobic amino acids immediately preceding the stop muscle, transmembrane N-CAM isoform which exhibits codon. A similar hydrophobic C-terminal tail has been dem- strong homology with a corresponding chick brain cDNA Table 1. Adhesion molecule families
+
1988
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Fig. 1. Fitll-length arnirio acid sequerice of a muscle-specific N-CAM isoforrn predictedfrom cDNA sequerice analyses This protein is most likely associated with the plasma membrane via phosphatidylinositol linkage. The structure can be summarized as follows. First, it has a signal peptide (amino acids 1-19); secondly, there is a large extracellular coding sequence which is believed to fold to form a structure having five repetitive disulphide loops. The cysteine residues that are believed to be involved in forming the C 2 domains are circled. In addition, the probable sites of carbohydrate linkage are shown as 0 . The muscle-specific sequence in this particular protein (amino acids 598-635) is shown by double underlines. Finally, the membrane-associated region (amino acids 742-76 1)is the site where phosphatidylinositol linkage occurs. Vol. 16
460 encoding N-CAM-140 (Murray et al., 1986). The nature of the remaining extracellular coding region of the human muscle transmembrane protein isoform awaits further characterization of cDNA clones. The restriction maps of these further clones have some similarity with the 5' EcoRl fragment of 11, but from restriction mapping there appears to be some sequence heterogeneity at the 5' ends of the cDNA clones. To ascertain the mRNA transcript from which each cDNA clone was derived a Northern blot hybridization analysis was undertaken. Both the transmembrane and non-transmembrane probes hybridized to mRNA transcripts of 6.7, 5.2, 4.3 and 2.9 kb from a mixed human myoblast/myotube culture and of 7.2 and 6.7 kb from human embryonic brain tissue. Both probes failed to hybridize to any RNAs from human fibroblasts or lymphoblastoid cells. To relate cDNAs to the RNA transcripts of origin, specific subfragment probes were utilized. The transmembrane, 3' terminal fragment of clone A4.4 hybridized specifically to 6.7 and 6.7 and 7.2 kb transcripts from muscle and brain, respectively, whereas the 3' untranslated fragment of the full length clone hybridized to muscle transcripts of 5.2, 4.3 and 2.9 kb. Thus N-CAM transmembrane isoforms in muscle are encoded by a 6.7 kb transcript and by 6.7 and 7.2 kb transcripts in brain tissue. A probe derived from the MSDl region only hybridized to non-transmembrane N-CAM isoforms from muscle cultures (5.2, 4.3 and 2.9 kb) and MSDl was not expressed in any additional mouse brain o r human brain transcript. The expression of MSDl appears to be specific to muscle and its expression is restricted to myotubes, myofibres and denervated myofibres. The isolation of cDNA clones encoding the full-length amino acid sequence of a human skeletal muscle non-transmembrane N-CAM isoform will now enable the development of expression models in vitro. Through study of these systems, the functional role of homophilic binding in myotube formation, innervation and reinnervation following neural injury may be clarified and in particular the role of the additional extracellular domains in muscle development may be defined. This work was supported by the Muscular Dystrophy Group of Great Britain and the Wellcome Trust. F.S.W. is a Wellcome Trust Senior Lecturer. Barthels, D., Santoni, M-J.. Wille, W., Ruppert, C., Chaix, J-C., Hirsch, M-R., Fonteilla-Camps, J-C. & Goridis, C. (1987) EMBO J. 6,907-914
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Molecular heterogeneity and differential expression of multiple protein kinase C subspecies in central nervous tissue MARK S. SHEARMAN: KATSUHIKO ASE: NAOAKI SAITO,? KAZUO SEKIGUCHI,* KOUJl OGITA,* USHIO KIKKAWA,* CHIKAKO TANAKAt and YASUTOMI NISHIZUKA* Department of *Biochemistry and t Pharmacology, Kobe University School of Medicine, Kobe 650, Japan Activation of protein kinase C (PKC) as a result of signalinduced diacylglycerol production, or directly, by exposure to synthetic permeable diacylglycerols or to tumour-promotAbbreviations used: PKC, protein kinase C; cDNA, complementary DNA.
ing phorbol esters, has been implicated in the regulation of cell secretion, the modulation of membrane conductance, functional modification of receptors and other components of the signal transduction machinery, and the control of gene expression (reviewed by Nishizuka, 1984, 1986). PKC was purified to apparent homogeneity, and considered to be a single molecular entity until recent molecular cloning analysis predicted the enzyme to exist as a family of multiple subspecies, having closely related structures (Parker et al., 1986; Coussens et al., 1986; Ono et al., 1986a,b; Knopf et al., 1986; Makowske et al., 1986; Ohno et a/., 1987; Housey et al., 1987). The relationship of the complementary DNA (cDNA) clones isolated in this laboratory to those of the 1988
