1. Protein Domains That Interact with Receptor Tyrosine Kinases: Structural Aspects Ming-Ming Zhou, Ph.D. MSSM Department of Pharmacology Course on “Cell Signaling Systems” January 27, 2005

2. The Role of Protein Domains in Cell Signaling PLC-  Sos Raf PI(3)K MEK MAPK c-fos, c-myc, c-jun Grb2 Shc P PTB SH2 CH P P P P P P RTK Growth Factors Ras GDP GDP Ras GTP SH2 SH3SH3

3. -pY-X-X-hy- SH2 -hy-X-N-P-X-(p)Y-Phospholipids PTB PH SH3WW EH 14-3-3 -N-P-F--R-S-X-pS-X-P- -P-X-X-P-X--P-P-X-Y- PDZ -E-S/T-D/V- COOH -... Protein Domains in Cell Signal Transduction

4. Human TrkA, pY490 HIIENPQpYFSDA 280 0.053 Mouse Polyoma MT, pY250 SLLSNPTpYSVMRS 320 0.140 Human ErbB3, pY1309 SAFDNPDpYWHSRLF 230 0.020 Human ErbB2, pY1248 AENPEpYLGLDV 1290 N.D. Human EGFR, pY1086 GSVQNPVpYHNQPLN 220 5.30 Human EGFR, pY1148 SLDNPDpYQQDFF 1000 0.200 Human EGFR, pY1114 TAVGNPEpYLNTVQ 290 0.140 Human EGFR, pY992 VADADEpYLIPQQ 130 680 Human EGFR, pY1173 TAENAEpYLRVA 60 770 Human TCR  Chain, pY141 GHDGLpYQGLSTATK 50 >10000 Protein pTyr Sites pTyr Peptides Shc SH2Shc PTB K d (  M) Ligand Recognition of the Shc PTB and SH2 Domains Ref: Zhou et al (1995) JBC 270, 31119-31123.

5. Phosphotyrosine Binding Domains  Ligand Specificity  Structural Versatility  Functional Diversity  SH2 Domain: pYxx  ---pYxx  ---  PTB Domain:  xNPxpY ---  xNPxpY--- Q:

6. 3D Structue of the Shc SH2 Domain/TCR Peptide Complex TCR  chain peptide: GHDGLpYQGLSTATK BG BC AA BB N C pY pY+3 EF  D’ EE FF CC BB DD GG AA R  B5 BG   N C pY pY+3  FF CC  DD GG  R  B5 R  A2 V  C3 H  D4 L  D6 A BG7 L  D5 T  E4 I BG4 Ref: Zhou et al (1995) PNAS 92, 7784-7788.

7. BG AA BB N C  D’ EE FF CC BB DD GG AA pY pY+3 DE pY pY+3 BG DE EE FF  D’ pY+3 pY BG DE EE FF  D’ pY pY+3 BG DE EE FF  D’ ShcLck Syp PLC-  1 Structures of SH2 Domains

8. Shc Lck Syp PLC-  1 Structures of SH2 Domains

9. 3D NMR Structure of the Shc PTB Domain/TrkA Complex Ref: Zhou et al (1995) Nature 378, 584-592.

10.  XNPXpY Recognition by the Shc PTB Domain

11. PTB Domain versus SH2 Domain Shc PTB Domain Shc SH2 Domain

12. Biological Ligands of PTB Domains PTB DomainBinding Partners Binding Sites Shc hTrkA, pY490 HIIENPQpYFSDA mPolyoma MT, pY250 SLLSNPTpYSVMRS hErbB3, pY1309 SAFDNPDpYWHSRL hEGFR, pY1148 ISLDNPDpYQQDFF IRS-1 hIR, pY960 LYASSNPEpYLSAS hIL-4R, pY497LVIAGNPApYRSFS hIL-4R, pY497 LVIAGNPApYRSFS hIGFIR, pY950LYASVNPEpYFSAS hIGFIR, pY950 LYASVNPEpYFSAS Numb LNX GLDNPAYTSSV NAK GFSNMSFEDFP Peptide Library YIGPYL X11, Fe65, Ced-6  APP GYENPTYKFFE

13. 3D Structure of the IRS-1 PTB Domain/IL-4R Complex Ref: Zhou et al (1996) Nature Structural Biology 3, 388-393.

14. Ligand Recognition of the IRS-1 PTB domain

15. Distinct Mechanisms for NPXpY Recognition IRS-1 PTB Domain Shc PTB Domain IRS-1 PTB Domain Shc PTB Domain

16. PTB & PH Domains: A Conserved Structural Fold ABCD

17. FGFRs (juxtamembrane region): FGFRs (juxtamembrane region): KSDFH SQMAV HKLAK SIPLR RQVTS NGFRs : NGFRs : TrkA, pY960 HIIENPQpYFSDATrkA, pY960 HIIENPQpYFSDA TrkB PVIENPQpYFGITTrkB PVIENPQpYFGIT TrkC PVIENPQpYFRQGTrkC PVIENPQpYFRQG RET pY1062 RET, pY1062 TWIENKLpYGRIS SNT/FRS2 Binding Sites in Neurotrophic Receptors

18. 1111 3 4 5 6 7 FGFR C N N C 2 1 8 ’’’’ 1111 3 4 5 6 7 FGFR C N N C 2 1 8 ’’’’ 3D Structure of the SNT-1 PTB Domain/FGFR1 Complex Ref: Dhalluin et al (2000) Molecular Cell 6, 921-929.

19. SNT-1 versus IRS-1 PTB domains R78 FGFR 1111 3 4 8 5 6 7 C N 2 1 R63 ’’’’ N C R212 IL-4R 1111 3 4 5 6 7 C N 2 1 R227 N C pY-3 pY-6 pY-8 pY ABAB SNT-1 IRS-1

20. Sequence Alignment of the SNT & IRS PTB Domains

21. R78 1111 3 4 8 5 6 7 FGFR C N N C 2 1 R63 ’’’’ Vector SNT-1 PTB Domain Domain SNT-1 PTB SNT-1 PTB Domain  8 His -, Leu -, Trp - Leu -, Trp - His -, Leu -, Trp - Leu -, Trp - His -, Leu -, Trp - Leu -, Trp - TRKBhFGFR1Vector SNT-1 Truncation Effect on FGFR & TRK Binding w/FGFR Yeast Two-Hybrid Binding Assay 1111 3 4 5 6 7 N 2 1 pY pY-5 R78 R63TrkBC C w/TrkB

22. Unifying Features of PTB Domains Ref: Yan & Zhou (2002) FEBS Lett. 513, 67-70.

23. Structure & Function of the SH3 Domain Ref: Zarrinpar (2003) Sci. STKE 2003(179), re8 CA B

24. RTKs: Activation by Receptor Dimerization Ref: Hubbard, et al (1998) JBC 273, 11987-11990.

25. Autoinhibition of Insulin and FGF Receptors IRKFGFR1K Unphosphorylated IRK (IRK0P) Tris-phosphorylated IRK (IRK3P) Unphosphorylated FGFR1K Tris-phosphorylated IRK (IRK3P) - Cis-inhibition & Trans-activation

26. AutoRegulation & Activation of Src Tyrosine Kinase

27. 3D Structure of Protein Tyrosine Kinase Hck Ref: Xu, et al (1997) Nature 385, 595-602; Sicheri, et al (1997) Nature 385, 602-609.

28. Rational Design of Nonpeptide Inhibitors of Src SH2 Domain Ref: Sundaramoorthi et al (2003) Biopolymers 71, 717-729.

29. 3D Structure of the Abl Kinase Domain/Drug Complex Ref: Daub, H. et al (2004) Nature Review Drug Discovery 3, 1001-1010.

30. Summary Structure-Function of Protein Domains in RTKs  Conserved 3D structural fold with divergent sequences  Conserved structural fold with different functions  Single protein domain with multiple functions  Cooperative regulation of RTK activity in cell signaling