The divergent receptors for TNF

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the production of diacylglycerol, which can then activate protein kinase C and also be metabolized to arachidonic acid 5. Workers who have attempted to distinguish the pathways through which free arachidonic acid is generated have tended to emphasize the importance of the phospholipase A2 and the phospholipase C pathways despite, in many cases, a lack of evidence that these are the major routes of arachidonic acid generation. Particular care needs to be taken in interpreting data as showing receptor- or calciumdependent activation of phospholipase A2 from experiments measuring an increase in release of free radiolabelled arachidonic acid from lipids prelabelled by incubation with radiolabeUed arachidonic acid, since this could have been generated through any of the routes shown in Fig. 1. Pinpointing the pathways that are involved in generating increased release of free arachidonic acid requires more stringent analysis of the membrane phospholipids that are hydrolysed, to determine which phospholipases have been activated. It has been suggested 6 that regulation of free arachidonic acid levels could also involve changes in the rate of reincorporation (reacylation) of arachidonic acid. There is some evidence; that

the reacylation pathway is regulated by protein kinase C. Treatment of platelets with phorbol esters or synthetic diacylglycerol leads to an increase in arachidonic acid release, and a decrease in arachidonic acid uptake by the cells. The effect of activation of protein kinase C on arachidonic acid was attributed to an inhibition of the activity of both the arachidonyl CoA synthetase and the acyltransferase (acylCoA"1-acylglycero-3-phosphoglyceride O-acyl transferase) involved in reacylation. Both of these enzyme activities were reduced in platelet homogenates after pretreatment of intact cells with protein kinase C activators;. Therefore, an alternative pathway for an increase of free arachidonic acid could involve inhibition of reacylation, following diacylglycerol production by phospholipases C or D, and protein kinase C activation. Production of arachidonic acid by this mechanism would clearly be independent of the metabolism of diacylglycerol, which is usually assumed to underlie arachidonic acid production via phospholipase C. Arachidonic acid and its metabolites play key roles in cellular regulation, and it is now becoming clear that the level of free arachidonic acid and its availability for eicosanoid synthesis can be

!tkt Enzymologists have long known that the same reaction may be catalysed by different enzymes (e.g. trypsin and subtilisin) that have little more in common than their active site machinery. Conversely, a single structural motif common to all immunoglobulins serves as the recognition platform for the endless variety of antigens encountered. A series of papers recently appearing in Cell 1,2 and S c i e n c e a show that at least one growth-factor receptor family may take advantage of both paradigms. Together, the work from three laboratories demonstrates that there are at least two receptors for tumor necrosis factor (TNF). Remarkably, the two receptor molecules are homologous only within a 150 residue cysteine-rich segment presumed to be the ligandbinding domain. An analog of this sequence is found also in the receptor for nerve growth factor (NGFr), a 366

¸ (ii

regulated in many ways. Arachidonic acid levels can be modified by changing the extent of its provision via the LDL receptor pathway, by phospholipase activity and by protein kinase C regulation of reacylation. There are also interactions between all of the pathways shown in Fig. 1 (including the ability of arachidonic acid itself to activate protein kinase C [Ref. 8]) which, despite its complexity, remains a simplified picture of the mechanisms involved in the control of arachidonic acid levels. References 1 Needleman, P., Turk, J., Jakchick, B. A., Morrison, A. R. and Lefkowith, J. B. (1986) Annu. Rev. Biochem. 55, 69-102 2 Smith, W. L. (1989) Biochem. J. 259, 315-324 3 Habenicht, A. J. R., Salbach, P., Goerig, M., Zeh, W., Janssen-Timmen,U., Blattner, C., King, W. C. and Glomset, J. A. (1990) Nature 345, 634-636 4 Axelrod, J., Burch, R. M. and Jelsema, C. L. (1988) Trends Neurosci. 11, 117-123 5 Exton, J. H. (1990) J. Biol. Chem. 265, 1-4 6 Irvine, R. F. (1982) Biochem. J. 204, 3-16 7 Fuse, I., Iwanaga,T. and Tai, H-H. (1989) J. Biol. Chem. 264, 3890-3895 8 McPhail, L. C., Clayton, C. C. and Snyderman,R. (1984) Science 224, 622-625

ROBERT D. BURGOYNE AND ALAN MORGAN MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK.

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hormone which is unlikely to have any structural resemblance to TNE Thus, the surface of the binding scaffold presented by this cysteine-rich fold is potentially complementary to a variety of ligands. Tumor necrosis factor is the collective name for two related cytokine hormones, TNF~ and TNF-[I. TNF-~ is named for its ability to induce hemorrhagic necrosis of certain routine tumors and its cytotoxicity to some tumor cell lines 4. Known also as cachectin, TNF~ is the primary mediator of inflammation, sepsis-induced shock and the wasting syndrome (cachexia) associated with chronic infection and

neoplastic disease. A multipotent cytokine, TNF-~ acts as an immunoregulator, represses lipogenic pathways in adipocytes, stimulates coilagenase production in epithelial cells and is a growth factor for fibroblasts. TNF-~, or lymphotoxin, is a lymphocyte-derived homolog of TNF~ and appears to effect many of the same functions. Consistent with its wide-ranging activities, receptors for TNF are expressed by all somatic cell types tested, with the exception of red blood cells. Receptors bind both TNF~ and TNF-~ (Ref. 5), but no other cytokines or growth factors, with dissociation constants in the nanomolar range. Recently, it has become evident that there are at least two molecular species of TNF receptor (TNFr), a high affinity (Kd = 0.07 riM) 75-80 kDa myeloid cell type receptor and a 55-60 kDa receptor of epithelial origin with an affinity

(~ 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00

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constant (Kd) of 0.3 nM (Ref. 6). The two molecules differ in glycosylation, yield different peptide fragments upon tryptic digestion and are immunologically distinct. Multiple receptor forms were also anticipated by earlier discoveries of TNF-binding proteins isolated from urine 7-9, and from the serum of cancer patients. Two such molecules, named TBP I and TBP I1 by Englemann et al. 7, have molecular masses in the 30 kDa range but differ in their N-terminal sequences. These binding proteins recognize TNF-~ and TNF-[3 with affinities comparable to those of the receptors themselves. The view that binding proteins are, in fact, shed extracellular receptors received support from experiments showing that incubation of cells with antisera to TBP I and II inhibited TNF binding. The TNF receptor cloned by Loetscher et al. 2 (from a human placental lambda gt11 cDNA library) using a PCR probe derived from the N-terminus of the 55 kDa TNF receptor, is identical to that cloned by Schall et al. ~ (from both placental and premyelocytic HL-60 cDNA libraries) using probes derived from TNF-binding protein sequences. This 'Type-l' 455 residue protein can be putatively subdivided into four domains: a signal sequence; a 182 residue extracellular cysteine-rich domain; a 20-22 residue transmembrane helical segment; and a 221-223 residue intracellular domain. The core of the cysteine-rich domain which is a fourfold inexact repeat of a 40 residue sequence - shows 34% amino acid sequence identity to the extracellular domain of NGFr~°. The homology derives primarily from the preserved registration of cysteine residues among the aligned sequences (see Fig. 1). In contrast, there is little significant homology between the cytoplasmic domains of TNFr and NGFr. On the basis of Scatchard analysis of radioiodinated TNF binding to recombinant receptor that is transiently expressed in COS cells, Schali et al. report high and low affinity sites with dissociation constants of 0.66rim and 20 riM, respectivei/. In contrast, Loetscher et al. 2 infer only a single class of binding site (Kd= 0.5 riM). Smith et alp discovered a very different breed of TNF receptor by expression screening a human lung fibroblast cDNA library. This 461 residue 'Type II' transmembrane protein also comprises signal, cysteine-

rich, transmembrane vCpqgkyihp--qnnsiCCtkChlgtylyndCpgpgqd-tdCr and cytoplasmic doss mains. The fourfold NG aCptglyths--ge---CCkaCnlgegvaqpCga--nq-tvCe kCgghdy ..... ekdglCCasChpgfyasrlCgp--gsntvCs cysteine-rich repeat T2 80 tCrlreyy---dqtaqmCCskCspgqhakvfCtk--tsdtvCd resembles most closely the sequen55 eC-esgsftasenhlrhClsCs-kCrkemgqveissC-tvdrdtvCces found in an NG pCldsvtfsdvvsatepCkpCt-eCvgl--qsmsapC-veaddavCr open reading frame T2 pCedgtftastnhapa-CvsCrgpCtgh--isesqpCdrthdr-vCof the Shope 8o sCedstytqlwnwvpe-ClsCgsrCssd--qvetqaC-treqnriCfibroma virus and the CD40 cell-surs~ gCrknqyrhy-wsenlfqCfnC---slClng-tvhlsCqekqntvC-C---aygyy-qdettgrCeaC---rvCeagsglvfsCqdkqntvCe face antigen from ~G nC---stgnycllkgqngCriCapqtkCpagygvsgh-tragdtlCe B cells, which is T2 tC---rpgwycalskqegCrlCaplrkCrpgfgvarpgtetsdvvCk also known as B80 lymphocyte actitChagfflr--ene---CvsCsnCkksl eCtk IC vation molecule 5s eCpdgtysdeanhv-dpClpCtvCedterqlreCtrwa-daeCe (Bp50); there is also NG T2 kCpphtysdslsptercgtsfnyisvgfnlypvnets ..... Ct some homology to pCapgtfsnttsst-diCrphqiCn .... vvaipgnasmdavCt 8o NGFr. The surprise is that the Type II Figure 1 receptor shows no Aligned cysteine-rich repeats of TNF receptors (TNFr) and more homology to related proteins (after Refs 1 and 2): 55, 55 kDa TNFr (Type I); the Type 1 TNFr NG, human nerve growth factor receptor; T2, open reading (less than 25% overframe of Shope fibroma virus; 80, 80 kDa TNFr (Type II). all sequence identity when aligned using the program, GAPn), than to the likely that these activities are mediated other members of the family. The by different receptors. The cytoplasmic cytoplasmic domain of the Type II domain of the Type l receptor contains receptor is 50 residues smaller than potential phosphorylation sites for that of Type I. The 57 residues cAMP-dependent kinase, protein kinase upstream of the transmembrane region C and tyrosine kinases, whereas the are rich in threonine, serine and proline Type If sequence does not. Neither residues, characteristic of O-linked intracellular domain bears any glycosylation sites. Affinity cross- resemblance to tyrosine kinases or the linking of the Type II receptor by intracellular sequence of other radioiodinated TNF-~ or -[~ shows a receptors. single species characteristic of the 80 The function and evolution of kDa receptor. Five residues (VAFTP) the TNF/NGF-binding domain is an just downstream of the putative signal intriguing structural problem. Schall et peptidase cleavage site are identical to aL suggest that the cysteine repeats the N-terminal residues of TBP lI (Ref. form a general factor-binding frame6). Thus, each of the TNF-binding work within which the peptide proteins has a receptor counterpart. sequence confers ligand specificity. The Whether the two TNF receptors overall charge of the repeat domain is diverged from a common ancestor or positive for both TNF receptors but arose by recombination events will negative for NGFr, suggesting that perhaps remain for some time a electrostatic complementarity may be challenging problem in molecular a component of specificityL3. Multiple evolution; Type I (55 kDa) TNFr/NGFr, cysteine-rich repeats in the putative and Type II (80 kDa) TNFr/CD40 binding domain, also a characteristic of antigens appear to be two distinct the LDL receptor TM, might function to branches of the family (Fig. 1). The optimize the interaction with TNF, exciting structural and functional which forms tightly packed trimers issues, however, can now be addressed in the crystal ~7,~8. Alternatively, the with the recombinant proteins in hand. complex array of species generated by TNF appears to activate a bewildering receptor/TNF crosslinking experiments ~9 array of signaling pathways, involving are equally consistent with the increased GTP-binding and hydrolytic formation of multiple receptor-TNF activity ~2, factor-dependent protein complexes at the cell surface. phosphorylation ~3 as well as the The newly discovered homologies induction of transcriptional activators among the receptors for TNE NGF and c-los and c-mycTM and transcriptional other less well characterized species factors binding to NF-~cB sites ~5. It is establishes a novel and very diverse .

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family of signaling molecules. The isolation and expression of these receptors should lead rapidly to definitive studies of their individual roles in cellular regulation, which may range as broadly as the versatile binding repeats they share.

References 1 Schall, T. J., Lewis, M., Koller, K. J., Lee, A., Rice, G. C., Wong, G. H. W., Gatanaga,T., Granger, G. A., Lentz, R., Raab, H., Kohr, W.J. and Goeddel, D.V. (1990) Cell 61, 361-370 2 Loetscher, H., Yu-Ching, E.P., Lahm, H-W., Gentz, R., Brockhaus, M., Tabuchi, H. and Lesslauer, W. (1990) Cell61, 351-359 3 Smith, C. A., Davis, T., Anderson, D., Solam, L., Beckmann, M.P., Jerzy, R., Dower, S. K., Cosman, D. and Goodwin, R. G. (1990) Science 248, 1019-1022 4 Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57,505-518

5 Stauber, G. B. and Aggarwal, B. B. (1989) J. Biol. Chem. 264, 3573-3576 6 Brockhaus, M., Schoenfeld, H-J., Schlaeger, E-J., Hunziker, W., Lesslauer, W. and Loetscher, H. (1990) Proc. Natl Acad. Sci. USA 87, 3127-3131 7 Engelmann,H., Novick, D. and Wallach, D. (1990) J. Biol. Chem. 265, 1531-1536 8 Seckinger, P., Isaaz, S. and Dayer,J-M. (1989) J. Biol. Chem. 264, 11966-11973 90lsson, I., Lantz, M., Nilsson, E., Peetre, C., Thysell, H., Grubb, A. and Adolf, G. (1989) Eur. J. Haematol. 42, 270-275 10 Johnson, D., Lanahan, A., Buck, C. R., Sehgal, A., Morgan, C., Mercer, E., Bothwell, M. and Chao, M. (1986) Cell47, 545-554 11 Software from the University of Wisconsin Genetics Computer Group: Devereux,J. R., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387 12 Imamura, K., Sherman, M. L., Spriggs, D. and Kufe, D. (1988) J. Biol. Chem. 263, 10247-10253 13 Schfitze, S., Scheurich, P., Pfizenmaier,K. and

Kr6nke, M. (1989) 1 Biol. Chem. 264, 3562-3567 14 Lin, J-X. and Vilcek, J. (1987) J. Biol. Chem. 262, 11908-11911 15 Duh, E. J., Maury, W. J., Folks, T. M., Fauci, A. S. and Rabson, A. R. (1989) Proc. Natl Acad. Sci. USA 86, 5974-5978 16 SOdhof,T. C., Goldstein, J. L., Brown, M. S. and Russell, D. W. (1985) Science 228, 815-822 17Jones, E. Y., Stuart, D. I. and Walker, N. P. C. (1989) Nature 338, 225-228 18 Eck, M. J. and Sprang, S. R. (1989) J. Biol. Chem. 264, 17595-17605 19 Smith, R. A., and Baglioni, C. (1989) J. Biol. Chem. 264, 14646-14652

STEPHEN R. SPRANG Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.

OBITUARY On 12 May 1990 in his office at Kyoto University, Japan, Professor Takashi Murachi died from a heart attack that occurred so suddenly that even his students working in the laboratory next door were not aware of it. He leaves his wife and two grown children. With his passing, the international scientific community has lost one of the foremost biochemists and scientific ambassadors of Japan. Takashi Murachi was well known throughout the world for his studies on thiol proteases, particularly stem bromelain and the calpains (also known as calcium-activated neutral proteases). The isolation and characterization of the calpains and their inhibitors (calpastatins) has been the major focus of his work during the past decade. He and his co-workers isolated these enzymes from various tissues, and applied the most modern methods of protein chemistry, enzymology and molecular biology to elucidate their structure, function and regulation. Although the precise physiological functions of these enzymes are not known, they can degrade various cytoskeletal proteins, including several of the high molecular weight microtubule-associated proteins, actin-binding proteins and various intermediate filaments. This suggests that they may play a role in the assembly and regulation of general cytoskeletal proteins. 368

They have also been suggested to be involved in memory development and are apparently largely responsible for the neuronal degeneration associated with stroke and possibly various senile dementias. As such, they are of pronounced importance to such pathologies as Alzheimer's and Parkinson's diseases. In addition, Professor Murachi

also published several articles on the use of enzymes as diagnostic reagents, as well as on growth factors and hormones. He was the author of over 200 articles published in prestigious journals both in Japan and in Western countries.

Takashi Murachi was born on 19 September 1926 in Kyoto, where he received the degree of Doctor of Medical Sciences in 1957. From 1957 to 1959 he was a recipient of a fellowship from the Rockefeller Foundation to work as a post-doctoral fellow on stem bromelain in my laboratory at the University of Washington. He returned to Japan in 1959, as Associate Professor of Biochemistry at Nagoya City University, and served as Chairman and Professor from 1966 to 1973. In 1974, he returned to his home in Kyoto, becoming Professor and Chairman of the Department of Clinical Science and Laboratory Medicine at Kyoto University Faculty of Medicine, and Director of the Central Chemical Laboratory at Kyoto University Hospital. He retired from this post on 31 March this year, and was in the process of establishing the Murachi Laboratory at the UNITIKA Research Center in Uji City, near Kyoto. Takashi Murachi received numerous honors. He was Secretary and Member of the Committee of Biochemistry of the Science Council of Japan, Past President of the Japanese Biochemical Society, President of the Federation of Asian and Oceanic Biochemists, and Honorary Member of the American Society of Biochemistry and Molecular Biology. He was serving as Chair of the Nominating Committee of the IUB at the time of his death. He was the recipient

© 1990,ElsevierScience Publishers Ltd,(UK) 0376-5067/90/$02.00