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Acknowledgements The author is grateful to F. Ali-Osman and M. T. Kuo (M.D. Anderson Cancer Center) for valuable discussions and critical reading of this manuscript.
References 1 Endicott, J. A. and Ling, V. (1989) Annu. Rev. Biochem: 58, 137-171 2 Ishikawa, T. (1990) Trends Biochem. Sci. 15, 219-220 3 Sies, H. and Katterer, B., eds (1989) Glutathione Conjugation, Academic Press 4 Hayes, J. D., Pickett, C. B. and Mantle, T. J., eds (1990) Glutathione S-transferases and Drug Resistance, Taylor and Francis 5 Kobayashi, K., Sogame, Y., Hara, H. and Hayashi, K. (1990) J. Biol. Chem. 265, 7737-7741 6 Kitamura, T. et al. (1990) Proc. NatlAcad. Sci. USA 87, 3557-3561 7 Akerboom, T. P. M., Narayanaswami, V., Kunst, M. and Sies, H. (1991) J. Biol. Chem. 266, 13147-13152 8 Fernandez-Chea,J. et al. (1992) J. Biol. Chem. 264, 17343-17348
9 Kondo, T., Murao, M. and Taniguchi, N. (1982) Eur. J. Biochem. 125, 551-554 10 LaBelle, E. F., Singh, S. V., Srivastava, S. K. and Awasthi, Y. C. (1986) Biochem. J. 238,443-449 11 Ishikawa, T. (1989) J. Biol. Chem. 264, 17343-17348 12 Bilzer, M. et al. (1984) Eur. J. Biochem. 138, 373-378 13 Sambamurti, K. et al. (1988) Genetics 120, 863-873 14 Hsu, I. C. et al. (1991) Nature 350, 427-428 15 Bressac, B., Kew, MI, Wands, J. and Ozturk, M. (1991) Nature 350, 429-431 16 Hayes, J. D., Judah, D. J., Mclellan, L. I. and Neal, G. E. (1991) Pharmacol. Ther. 50, 443-472 17 Degan, G. H. and Neumann, H. G. (1978) Chem. Biol. Interact. 22, 239-255 18 Ozawa, N. and Guengerich, F. P. (1983) Proc. Natl Acad. Sci. USA 80, 5266-5270 19 Ishikawa, T., Kobayashi, K., Sogame, Y. and Hayashi, K. (1989) FEBS Lett. 259, 95-98 20 Samuelsson, B. et al. (1987) Science 237, 1171-1176 21 Bach, M. K., Brashler, J. R. and Morton, D. R. (1984) Arch. Biochem. Biophys. 230, 455-465 22 Sonderstr6m, M., HammarstrSm, S. and Mannervik, B. (1988) Biochem. J. 250, 713-718
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19764-19770 34 Atsmon, J. et al. (1990) Cancer Res. 50,
PROTEINSEQUENCE MOTIFS The human protooncogene ret: a communicative cadherin? The human ret protooncogene (c-ret) encodes a putative receptor tyrosine kinase (RTK) with a large extracellular domain 1, the expression of which is tissue and/or developmental stage-specific2. Oncogenic activation of this RTK is frequently observed in papillary thyroid carcinoma 3. Its normal functions and putative ligand(s) are as yet unknown, since analysis of the extracellular domain amino acid sequence did not reveal any structurally or functionally significant data. A comparative analysis of the c-ret amino acid sequence involving iterative repeat searches has now revealed the presence of regularly spaced, short repeats, suggesting the repetition of larger (-100 amino acids) protein modules in c-Ret (Fig. lc). A database search for proteins containing at least three repetitions of the consensus sequence derived from the short repeats (Fig. lc) has identified only members of the cadherin superfamily if o~e mismatch was allowed. Moreover, allowin~ an additional mismatch still failed to identify other proteins, which further supports the significance of this match of the putative c-ret structural profile with cadherins.
Cadherins are a superfamily of transmembrane proteins that mediate homophilic Ca2+-dependent cell-cell adhesion, which is important in differentiation, morphogenesis and tumor suppression 4. Their extracellular portion usually consists of four related domains of about 110 amino acids and one cysteine-rich domain of similar size. These domains contain the Ca2~-binding motifs 5 necessary for resistance to proteolysis and homophilic adhesion of cadherins. All known cadherin domains share a consensus sequence with clusters of highly conserved residues at the Ca2+binding sites and at another site of unknown function. Detailed analysis of the initially identified similarities betwen c-Ret and cadherins finally yielded several lines of evidence supporting their distant relationship. Most remarkably, the repetitive regions-identified in c-Ret are not only homologous in position and sequence to the cadherin Ca2+-binding sites, but they also coincide exactly with the best-conserved, hallmark residues of the cadherin superfamily. Moreover, the othe~r highly conserved motif of the cadherins (LDREXXXXYXL) is also found in an exactly corresponding, position in c-Ret (Fig. lb). This is strong independent evidence (as this motif was not used in the initial database search) for a relationship between c-Ret and the cadherins. To further assess the significance o f this relationship, the sequence profile of a
multiple sequence alignment of the most conserved parts (comprising the sequences ranging from the conserved LDRE motif to the putative Ca2+-binding motif at the end of each domain) of various cadherin domains 6'7 was searched against protein databases using the program PROFILESEARCH.8 This yielded a significant alignment score of cadherin domains with c-Ret being 7.53 (length normalized) standard deviations above the mean of scores with unrelated sequences. This clearly supports the assignment of c-Ret to the cadherin superfamily. This relationship is highlighted by several other features: first, both are cellular receptors with a single transmembrane and a large extracellular domain; second, the extracellular domain of both is built of repetitive units of similar size; third, the region best conserved between them, as judged by the program ALIGN9 (Fig. 1), is positioned at the same relative distance from the transmembrane domain; and fourth, both harbou r cysteine-rich domains close to the membrane. In summary the marked similarities suggest that c-Ret might exert homophilic Ca2+-dependent binding to a second c-Ret molecule exposed on another cell, which could trigger its tyrosine kinase activity, thus signaling specific cell-cell recognition. Several experimental observations support this proposition: the long-term induction of c-ret expression during retinoic acid-triggered © 1992, Elsevier Science Publishers, (UK)
TIBS 17 - N O V E M B E R 1 9 9 2
*WEKLyVdQaaGtP L L y V - ~ c *WEKLsVrNr-G fPLLtva2+-binding ?
l i :il \haLrOAPEe-VPSF Ca2+-binding
%DRExxxxYxL ! LDRE(]rekYeLI:::::::I
IxVxDxNDn-xPxF~ LxVxDxDxpxxPaW~ VxVxDvNN xPxF xxVxDxNDx-xPxF~
Cysteine-rich d o m a i n s
cadherins tend to aggregate at cell-cell contact sites, suggesting an activation mechanism for c-Ret that could be very similar to the ligandbinding/oligomerization/ trans-phosphorylation scheme known from other RTKs ~°. The c-Ret protein would not be the first case of an adhesion-triggered RTK. It was reported very recently that D-Trk, a Drosophila developmental RTK containing adhesive immunoglobulin-like modules similar to those found in the trk family of neurotrophin receptors u can promote homophilic, although Ca2+-independent, adhesion with concomitant activation of its kinase activitym. Thus, activation of tyrosine kinases by homophilic intercellular adhesion might be a general phenomenon in the signaling of cell-cell recognition events. These novel clues to the structural and functional organization of c-Ret not only suggest a role for c-Ret in differentiation and/or morphogenesis, but also may further our understanding of the mechanisms of its oncogenic activation.
Acknowledgement I am grateful to T. Etzold, EMBL, Heidelberg, for his help in database searches with multiple sequence alignments.
Tyrosine kinase domain
c-Ret Figure 1
Schematic representation of the sequence similarities and the topological analogies between c-Ret and cadherins. (a), (¢) Alignments, consensus sequences and relative positions of the repeated sequences of the putative Ca2+-bindingsites found in cadherins and c-Ret. The repeats in c-Ret were found by eye and by application of the programs LINEUP, PROFILE and PROFILEGAp8;similarities between individual pairs of repeats are extending further than shown in (c). Identical and similar residues (amino acid similarity groups: IVLMF, ASTGPC, RHK, EDQN, WYF) are shown in uppercase letters. Identical residues occurring in at least more than half of the repeats contribute to the consensus sequence. The shown c-Ret repeats start at positions 54, 163, 260, 296, 374, 465 and 607, respectively. (b) Boxes indicate putative modulesl shaded areas represent the regions best conserved between c-Ret and cadherins: program ALIGN (MD-matrix, bias = 6, penalty = 6) gives 7.8 SD units for comparing residues 202-386 of c-Ret and residues 133-320 of ' murine placental cadherin 13. Hatched boxes indicate the similar locations of cysteine-rich domains. Sequences written between the two receptors represent structural features identified independently of the initial database search. Asterisks: a different type of repeats found in the amino-terminal region of c-Ret confirming its repetitive character. Exclamation marks: positions and consensus sequences of highly conserved regions in cadherins (left), which are also found in c-Ret (right).
morphological differentiation of the neuroblastoma cell line SK-N-BE is accompanied by an extensive adhesive clustering of cells (G. Della Valle, pers.
References 1 Takahashi,M. et al. (1988) Oncogene 3, 571-578 2 Szentirmay,Z. et a/. (1990) Oncogene 5, 701-705 3 Grieco, M. et al. (1990) Cell 60, 557-563 4 Takeichi, M. (1991) Science 251, 1451-1455 5 Ozawa,M., Engel,J. and Kemler,R. (1990) Cell 63, 1033-1038 6 Mechanic,S., Raynor,K., Hill, J. E. and Cowin, P. (1991) Prec. Natl Acad. Sci. USA 88, 4476-4480 7 Mahoney,P. A. et al. (1991) Cell 67,853-868 8 Devereux,J., Haeberli,P. and Smithies, O. (1984) Nucleic Acids Res. 12,387-395 9 Dayhoff,M. 0., Barker,W. C. and Hunt, L. T. (1983) Methods Enzymol. 91, 524-545 10 UIIrich,A. and Schlessinger,J. (1991) Cell 61, 203-212 11 Schneider,R. and Schweiger,M. (1991) Oncegene 6, 1807-1811 12 Pulido,D. et al. (1992) EMBOJ. 11, 391-404 13 Nose,A., Nagafuchi,A. and Takeichi M. (1987) EMBO J. 6, 3655-3661
RAINER SCHNEIDER Institute of Biochemistryof the Universityof Innsbruck, Peter-Mayr-Strassela, A-6020 Innsbruck, Austria.
commun.), and furthermore, high levels of tyrosine phosphorylation have frequently been observed at cadherinmediated celt-cell contact sites 4. Also,