The gap junction proteins

The gap junction proteins

375 T I B S 11 - September 1986 Condusion Mammalian iron metabolism evolved to keep ionic iron largely sequestered in proteins4, 5, which probably p...

381KB Sizes 7 Downloads 157 Views

375

T I B S 11 - September 1986

Condusion Mammalian iron metabolism evolved to keep ionic iron largely sequestered in proteins4, 5, which probably provided an extracellular antibacterial mechanism and perhaps, via transferrin receptors, a control mechanism for cell growth 8. Metal sequestration is equally important in minimizing iron-dependent and copper-dependent (Table I) radical reactions, and should be regarded as a significant part of aerobic antioxidant defence. References i Wolff, S. P., Garner, A. and Dean, R.T. (1986) Trends Biochem. Sci. ll, 27-31 2 Yagi,K. (1986)Trends Biochem. Sci. 11, 18-19 3 Halliwell, B. and Gutteridge, J. M. C. (1985) Mol. Aspects Med. 8, 89-193 4 Octave, J. N., Schneider,Y. J., Trouet, A. and Crichton, R.R.(1983) TrendsBiochem. Sci. 8, 217-220 5 Crichton, R. R. (1984) Trends Biochem. Sci. 9, 283-:'.86 6 Halliwell, B. and Gutteridge, J. M. C. (1985)

Free Radicals in Biology and Medicine, Clarendon Press 7 Esterbauer, H. (1985)in FreeRadicals in Liver Injury (Cheesman,K. H., Dianzani,M. U. and Slater, T. F., eds), pp. 29-47, IRL Press 8 Weinberg, E. D. (1984) Physiol. Rev. 64, 65102 9 Gutteridge, J, M. C., Paterson, S. K., Segal, A.W. and Halliwell, B. (1981) Biochem. J. 199,259-261 I0 Baldwin,A., Jenny, E. R. and Aisen, P. (1984) J. Biol. Chem. 259,13391-13394 11 Halliwell,B., Gutteridge, J. M. C. and Blake, D. (1985) Phil. Trans. R. Soc. London B311, 659-671 12 McCord, J. M. (1985) New Engl. J. Med. 312, 159-163 13 Blake, D. R., Gallagher, P. J., Potter, A.R., Bell, M. J. and Bacon, P. A. (1984) Arthritis Rheum. 27,495-501 14 Doly, M., Bonhomme, B. and Vennat, J.C. (1986) OpthalmicRes. 18, 21-27 15 Gutteridge, J. M. C. (1986) FEBS Lett. 201, 291-295 16 Gutteridge, J.M.C.(1985)Biochim. Biophys. Acta 834, 144-148 17 Davies, K. J. A., Sevanian, A., MuakkassahKelly, S. F. and Hochstein,P. (1986)Biochem. J. 235,747-754

The gap junction proteins d. P. Revel, S. B. Yancey and B. d. I'ticholson Gap junctions are ubiquitous structures consisting o f paired transmanbrane channels which allow for celt-cell exchanges. While the morphological appearance and gweral physiologkal properties o f gap junctions are surprisingly uniform throughout the animal kingdom there is increasing evidence for the presence o f tissue-specific proteins, which are highly conserved species. There is evidence that these proteins may belong to several protein families,

Gap junctions were originally discovered in excitable tissues - as electrical synapses in brain and between adjacent heart and adjacent smooth muscle cells, So the description of electrical communication between adjacent non-excitable cells in Drosophila salivary glands I and squid embroyos 2 created quite a stir. It was eventually established that electrical coupling and gap junctions went hand in hand and were virtually ubiquitous 3. Gap junctions are now believed to be involved in diverse cellular activities, including metabolic cooperation4, 5 and the signaling involved in the control of growth and differentiation 6. Gap junctions consist of clusters of transmembrane channels, each of which is called a connexon. Connexons in neighboring membranes are paired, interacting end to end with those of the opposite cell membrane to form a c o m plete channel (Fig. 1). Ions and waterJ. P. Revel, S. B. Yanceyand B. J. Nicholson are at the Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

soluble low molecular weight cornpounds are thus allowed to pass between adjacent cells. In thin sections studied in the electron microscope, gap junctions are seen as regions where only a narrow gap separates the apposed membranes of coupled cells7. In freeze-fractured sampies, gap junctions are visualized as quasicrystalline arrays of membrane particles, each representing a connexon (see Refs 3 and 8). Nearly all models of gap junctions depict connexons as consisting of four to six protein subunits forming the cylindrical wall of the transmembrane aqueous channel 9-H (Fig. 2). With recent improvements in techniques for the bulk isolation of junctions from different tissues and the increasingly detailed analysis of their constituents, it is becoming evident that there is not just one but a multiplicity of junction proteins (see Ref. 12). It has been known for quite some time that junctions in arthropods differ very greatly, both morphologically and physiologically, from those in vertebrates. This difference is also reflected in their protein

18 Gutteridge, J. M. C., HalliweL1, B., Treffry, A., Harrison, P. M. and Blake, D. (1983) Biochem.J. 209, 557-560 19 O'Connell, M. J., Ward, R. J., Baum, H. and Peters, T. J. (1985)Biochem. J. 229, 135-139 20 Biemond, P., van Eijk, H. G., Swaak, A. J. G. and Koster, J. F. (1984) J. Clin. Invest. 73, 1576-1579 21 Young, S. P., Roberts, S. and Bomford, A. (1985) Biochem. J. 232,819--823 22 Floyd, R. A. (1983)Arch. Biochem. Biophys. 225,263-270 23 Cutler, R. G. (1984)in FreeRadicalsin Biology (Vol. 6) (Pryor, W. A., ed.), pp. 371-428, Academic Press 24 Gutteridge,J.M.C. andStocks, J.(1981)CRC Crit. Rev. Clin. Lab. Sci. 14,257-329 25 Martin, W., Loschen, G., Gunzler, W. A. and Hohe, L. (1985)Agents Actions 16, 48-49 26 Tamir, H. and Liu, K. P. (1982)J. Neurochem. 38, 135-141 27 Stocks, J., Gutteridge, J. M. C., Sharp, R. J. and Dormandy, T. L. (1974)Clin. Sci. 47,215222 28 Gutteridge, J. M. C., Richmond, R. and Halliwell, B. (1979)Biochem. J. 184,469--472 29 Wills,E. D. (1969)Biochem. J. 113,325-332 30 HalliweU,B. and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246,501-514 composition 13. The specialized physiological properties of junctions in different tissues of vertebrates (see Ref. 14) may reflect differences in junctional structure based, at least in part, on the presence of different junction proteins. The concept of diversity of the gap junction proteins was supported initially by our peptide mapping and the first results of amino acid sequencing 12. A t present, however, only proteins from the putative j unctions in three tissues - liver, heart and lens have been analysed in any detail. The connections between liver cells are among the best studied gap junctions. There is a great deal of support for a protein of 26-28 kDa as a major cornponent of rat liver junctions (see Ref. 12), although other molecular sizes have also been suggested in the literature. Antibodies raised against the 28 kDa polypeptide bind specifically to gap junctional membranes and recognize a 28 kDa component in several other tissues 15,16. Binding of antibody to the 28 kDa component blocks communication between cells, as expected if it were to interfere with junction permeabilityr,14,Z7. Although the 28 kDa polypeptide is p r o b a b l y a m a j o r j u n c t i o n c o m p o n e n t , it may not be the only one. In heavily loaded SDS gel electrophoresis lanes, other components are seen even in the cleanest gap junction fractions. Many of these extra bands can be shown to represent degradation and/or aggregation products of the 28 kDa protein ~8. The presence of a band migrating as a 21

~)1986, ElscvierS¢iencePublishersB.V.,Arnsterdam

0376 5G67/86/$02.00

376

T I B S I 1 - S e p t e m b e r 1986

(a) ~

@

@

~ o/ "~ ~ (

~ ( ~ (b)

Fig. 1. Diagram of a gap junction. (a) Two cell membranes (indicated by the gray stippling) are held in close apposition by connexons, which are arranged end to end to form a continuous aqueous channel from the cytoplasm of one cell to that of its neighbor. (b) Seen face on, as in freeze-fractured samples or negatively stained preparations, the con-

nexons are arranged in a fairly orderly array. The centralporeisinsulatedbythechannelwallfromthe intercellular space between connexons,

kDa component, however, cannot be explained in either of these ways. It represents 10% of the protein in the final fractions from rat and as much as 3040% in those from mouse ~s. Because this component does not change in concentration after partial hepatectomy ~9,while the 28 kDa component20 and morphologically detectable gap junctions do 8, the 21 kDa band was at first dismissed as a contaminant. However, sequence analysis of the 21 kDa protein shows it to be clearly homologous with the 28 kDa molecule (Nicholson et al., unpublished), and thus it might well be a true junction component. During solubilization of isolated gap junctions the 21 and 28 kDa proteins aggregate independently into homodimers only. There may thus be multiple junction molecules in the liver, two of which at least (21 and 28 kDa) belong to the same family. Although we only know about 10% or so of the sequence of several of the molecules under discussion, and that onlyin a few species, itis alreadybecoming apparent that these molecules are highly conserved (90-98% homology) between species. Both the 28 and 21 kDa proteins may form separate arrays, not distinguishable morphologically from each other (or at least not yet distinguished). Alternatively, there may be protein heterogeneity at the connexon level with different proteins making up a single connexon, or heterogeneity at the junctional plaque level, with connexons of different composition making up each junctional array,

Other entities proposed to be part of the channel are less likely to be so. A 54 kDa component, recognized in western blots by its immunological cross-reactivity with antibody raised against the 28 kDa protein6, has been suggested as a possible precursor. But as a cDNA clone for liver gap junction protein recently isolated by D. Paul contains stop codons which would limit the size of the encoded protein to a calculated mol. wt of 3200021, it is likely that the 5 4 k D a polypeptide represents a contaminant or an aggregation product. A 16 kDa protein 22 has been found to co-isolate with junctional plaques and changes concentration as gap junctions do, for example after hepatectomy. It is found in junction fractions of a variety of tissues and cell lines, but it is seen only under specific conditions of isolation, has no immunological cross-reactivity and has no sequence homology (at the amino terminus, or as reflected in peptide maps) with the 28 kDa component. The 16 kDa protein is apparently associated with junction fractions but it is not clear whether it represents an intrinsic junc, lion molecule. Only further sequencing will indicate whether it is related to the other two previously discussed junctional components or is a contaminant which is specially difficult to recognize as such. COl'lnO×oIq ~ ~ ~ ~

~ ~

~

~ I )

subunit~ Fig. 2. Hypothetical arrangement of protein subunits in a single connexon (see discussion in Ref.

30). Six subunits are arranged so as to form the wall ofa centralaqueous pore, extending through the cell membrane. Each subunit may be different in cornposition from its neighbors. At present, evidence points to two separate but related molecules in liver and only one in heart and in the structures believed to represent lens junctions. Each transmembrane element is indicated by a circle as if it were an a.helix in cross-sec~on. In this hypothetical model the pore

would be lined by six amphiphilic helices with their hydrophilic faces bordering the pore itself. The other transmembrane elements, interacting with the lipid components of the membrane, could be largely hydrophobic,

Less is known about the heart junction protein(s) than about those of the liver we have just described. The heart junclion protein originally isolated from rat and rabbit was described as molecule(s) of between 28 and 34 kDa. There were clear indications that these molecules were sensitive to proteases. Work by Manjunath and Page23 and in our laboratory (unpublished) now shows that a 2830 kDa component is a peptide formed by the a c t i o n o f a s e r i n e p r o t e a s e o n a precursor of 45-47 kDa. This large molecule and the smaller peptides seen earlier, as well as molecules of intermediate size, are all found to have the same amino terminus, with clear sequence homology to the liver junction proteins discussed previously. The 17 kDa moiety of heart that is lost during isolation must therefore represent the carboxy-terminal region of the native junction protein. Its disappearance coincides with the removal of a fuzzy, electron-dense cytoplasmic coat found in heart 23 (but not seen in liver). The presumption is therefore that all or at least part of the carboxy-terminal portion of the molecule is at the cytoplasmic face. It may be unique to the heart or at least to gap junctions in excitable tissues. There is as yet no evidence for other junction proteins in the heart. In lens fiber cells, at least two proteins have been discussed as part of the junction. One of these, the main intrinsic protein (MIP) is very abundant and is found in fractions enriched in junctions (see Ref. 24). The problem here is that the junctions are atypical in terms of morphological appearance and that many antibody preparations against MIP stain all membranes instead of junctional areas specifically (see Ref. 25). In fact, one laboratory has prepared several antibodies that stain all membranes except the junctional regionsz6. All of this gives pause for thought, although there is also data supporting the junctional nature of MIP 27. The other molecule proposed to constitute the lens fiber junctionzs has a M r of 70 000. The 70 kDa component is, however, present at relatively low concentrations (gels must be loaded heavily to detect it) although the lens cortex appears well endowed with gap junctions. Immunostaining shows the 70 kDa protein to be organized into small plaques, a morphological distribution expected of gap junctions. MIP has been sequenced by Gorin and Yancey29, and has a calculated mol. wt of 28 200, in line with the size of many other gap junction proteins. However, there is no common sequence yet detected between MIP and the liver or heart proteins and most

377

T I B S 11 - S e p t e m b e r 1 9 8 6

workers find no immunological cross-

there needs to be such apparent redun-

reactivity. Model building suggests that MIP has at least one amphiphilic segment, as in the case of other channelforming proteins (Fig. 2; Ref. 30). Reconstitution experiments with MIP support a channel, possibly a cell-cell channel-like role, but are not conclusive

dancy even in a single tissue, such as liver.

because of impurities in the starting material. Accepting, for argument's sake, that either MIP or the 70 000 kDa protein of the lens fiber is truly a gap junction channel, one is led to the conclusion that there are specific junctional polypeptides in various tissues. The differences observed between junction polypeptides of various tissues may be related to physiological parameters. While some of the proteins (the 21 and 28 kDa liver proteins and those of heart) appear to be related by their amino acid sequence, others, such as the putative gap junction proteins of the lens (MIP) or the 16 kDa molecule discussed above, are not. Taken at face value the data available today would suggest several classes of polypeptides associated with gap junctions and therefore possibly involved in cell-cell communication. One must ask what the role of each might be, and why

15 Dermietzel, R., Liebstein, A., Frixen, U., Janssen-Timmen,V., Traub, O. and WiUecke, K. (1984)EMBOJ. 3, 2261-2270 16 Hertzberg, E. L. and Skibbens, R. V. (1984) Ce1139,61-69

References 1 Lowenstein, W. R. (1981) Physiol. Rev. 61, 829-913 2 Potter, D., Furshpan, E. and Lennox, E. (1966) Proc. Natl Acad. Sci. USA 55,328-335 3 Gilula, N. B., Reeves, R. O. and Steinbach,A. (1967)Nature 235,262-265 4 Pitts, J. D. (1980)In Vitro 16, 1049-1056 5 Hooper, M. L. and Subak-Sharpe,J. H. (1981) Int. Rev. Cytol. 69, 45-104 6 Warner, A. E., Guthrie, S. C. and Gilula, N. B. (1984)Nature 311,127-131 7 Revel, J. P. and Karnovsky, M. J. (1967) J. Cell. Biol. 33, c7-12 8 Meyer, D., Yancey, B., Revel, J. P. and Peskoff, A. (1981)J. Cell Biol. 91,505-523 9 Caspar, D., Goodenough, D., Makowski, L. andP~llips, W.(1970)J. CellBiol. 47,49~O

10 Unwin, P. N. T. and Zampighi, G. (1980) Nature 283, 545-549

11 Makowski, L. (1985) in Gap Junctions (Bennett, M. V. L. and Spray, D. C. eds), pp. 5-12, Cold SpringHarbor Laboratories 12 Revel, J. P., Nicholson, B. J. and Yancey, S. B. (1985)Annu. Rev. Physiol. 47,263-269 13 Finbow, M. E., Buultjens, T. E., Lane, N.J., Shuttleworth,S. and Pitts, J. D. (1984)E M B O y. 3, 2271-2278 14 Spray, D. C., White, R. L., Mazet, F. and Bennett, M. V. L. (1985) Am. J. Phys. 17, H753-H764

17 Hertzberg, E. L., Spray, D. C. and Bennett, M.V.L. (1985) Proc. NatlAcad. Sci. USA 82, 2412-2416 18 Henderson, D., Eibl, H. and Weber, K. (1979) J. Mol. Biol. 132,193-218 19 Traub, O., Druge, P. M. and Willecke, K. (1983)Proc. Natl Acad. Sci. USA 80, 755-759 20 Finbow, M., Yancey, S. B., Johnson, R. and Revel, J. P. (1980) Proc. Natl Acad. Sci. USA 77,970-974 21 Paul, D. (1985)J. Cell. Biol. 101,393a 22 Finbow, M., Shuttleworth,J., Hamilton, A. E. and Pitts, J. D. (1983) EMBO J. 1479-1486 23 Manjunath, C. K. and Page, E. (1985)Am. J. Phys. 248, H783-H791 24 Bloemendal, H. (1982) CRC Crit. Rev. Biochem. 12, 1-38 25 Fitzgerald P. G., Bok, D. and Horwitz, J. (1983)J. Cell. Biol. 97, 1491-1499 26 Paul, D. L. and Goodenough, D. A. (1983) J. Cell. Biol. 96,625-632 27 Sas, D., Sas, J., Johnson, K., Menko, A. and Johnson, R. (1985)J. Cell. Biol. 100,216-225 28 Kistler, J., Kirkland, B. and Bullivant, S. (1985)J. Cell Biol. 101,28-35 29 Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.P. and Horwitz, J. (1984)Cell39, 49-58 30 Revel, J. P. and Yancey, S. B. (1985) in Gap Junctions (Bennett, M. V. L. and Spray, D. C. eds), pp. 33-48, Cold Spring Harbor Laboratories

Reflections on biochemistry Early X-ray stu dies of a biopolymer R. D. Preston In these days of highly sophisticated and successful approaches, both physical and intellectual, to the investigation of the structure and function of biopolymers it may be salutory occasionally to look back at the earlier 'simpler' days when the study was in its infancy 1. Not so much in compassion for the simple treatment then possible - for we thought of ourselves as the sophisticates - but to recall the difficulties under which the earlier workers laboured and the molecular species which were of interest at the time and upon which the whole m o d e m edifice therefore stands. The term molecular biology has come to be almost synonymous with gene manipulation but this is not how it was originally founded and the first relevant ideas were not developed either with nucleic acids or proteins. As defined by Astbury, for instance, as late as 1950, molecular biology was ' . . . an approach from the viewpoint

of the so-called basic sciences with the leading idea of searching below largescale manifestations of classical biology for the corresponding molecular plan '2. In this looser sense, molecular biology began with a polysaccharide, cellulose, The precise moment at which a discipline is born is always difficult to define, but it could be argued that the seminal event was the use by Carl von NiigelP, 1858, of the cellulose of plant cell walls to develop the idea of micelles and to demonstrate the crystallinity of a natural structural material. From this time until the 1930s developments came almost solely through the study of cellulose, culminating in the finding in 1920 by Polanyi that cellulose yields an X-ray diagram and the determination by Sponsler and Dore (1926) 4 and by Meyer and Mark (1928) 5 of a unit cell. A major puzzle had been that cellulose, unlike its component glucose, is non-reducing. In

molecules,the absencemof a cthe r O -had concept c e ltherefore l u l of o s e been thought of as a dimer, or at least a cyclic structure of low molecular weight. This had been reconciled with the known high molecular weight of cellulose by supposing that these smaller units were tightly aggregated into the micelles of N~igeli and in this sense N/igeli's ideas had held up further progress. Once a unit cell was obtained, however, and the cellobiose residues positioned within it, the unavoidable conclusion was that cellulose is a long chain polymer, the first example of a linear macromolecule. This was the situation when J. H. Priestley invited me, as a raw physics graduate, to join his Department of Botany and, later, W . T . Astbury 6 offered me the use of his X-ray laboratory. The basis of the problem I defined at that time was twofold. Firstly, plant cell walls were anisotropic and this might derive from the orientation of the constituent cellulose crystallites. Secondly, the orientation of the crystallites and any changes in the orientation through the wall might reflect conditions at the cytoplasmic surface, giving one of the

1986, Elsevier Science Publishers B.V., Amsterdam

0376- 5667/86~02,1KI