1. Perturbation of Signal Transduction Pathways for Growth Factors and Growth Inhibitors in Malignant Transformation 3 2 1 - - 1 2 3

2. Different isoforms of PDGF form three types of receptor dimers PDGF-ABPDGF-BBPDGF-DDPDGF-AAPDGF-CC Ligand isoform Extra- cellular TM Intra- cellular Receptor type 

3. In vivo function of PDGF Embryonal development kidneys (mesangial cells) blood vessels (smooth muscle cells, pericytes) lungs (alveolar smooth muscle cells) CNS (oligodendrocytes) Stimulation of wound healing Regulation of interstitial fluid pressure

4. Ligand-induced activation of PDGFR  causes a conformation switch in the receptor C-terminus

5. 010203040506070 0 2500 5000 7500 10000 Wt ct29 ct46 ct75 Time (min) Kinase actvity 010203040506070 0 2500 5000 7500 10000 PDGFR  autoinhibition by a C-terminal motif No PDGF + PDGF Time (min) Kinase actvity PDGFR  ct75- ct46- ct29- - Y

6. Control PDGF Wtct46 -D850N 0 10 20 30 40 50 60 70 80 vector WT D850N ct46 ct46-D850N M-Ras Q71K Number of Foci PDGFR  activation by mutating the activation loop and the C-terminus

7. Binding of SH2 domain molecules to autophosphorylated tyrosines in the PDGF  -receptor P P P P P P P P P P P P P P P P P P P P P P P P PLC-  mitogenicity 562 579 581 589 716 740 751 763 771 775 778 857 966 970 1009 1021 SHP-2Grb2/Sos1RasErk MAPK mitogenicity STAT RasGAP Nck Src mitogenicity Shc Grb2/Sos1Ras Erk MAPK mitogenicity PI3’-kinaseRho, Rac, JNK MAPK actin reorganization and chemotaxis Grb2/Sos1RasErk MAPK mitogenicity SSSS SSSS Akt survival

8. 200 116 97 66 50 37 25 1 2 3 PDGFR  PDGFR  RasGAP / FAK Hsp84 Hsp A5 SHP2 Stam1 Erk1 Grb2 Alix Identification of tyrosine phosphorylated proteins after PDGF stimulation 1.Serum-starved cells 2.PDGFR  expressing cells stimulated with PDGF-BB 3.PDGFR  expressing cells stimulated with PDGF-BB

9. Alix overexpression decreases the rate of PDGFR  removal from the cell surface PAE/PDGFR  PAE/PDGFR  -Alix 0 50 100 0103060 PDGF-BB stimulation (min) Cell surface PDGFR  (% of initial) 1.0 8.2 Alix PAE/PDGFR  -Alix  -actin PAE/PGFR 

10. K Monoubiquitylation Ub KKK Multimono- ubiquitylation Ub Ubiquitylation of proteins K Polyubiquitylation (via K48 and K63) Ub K

11. 0 2 4 6 060120 Time (min) Ubiquitination (a.u.) PAE/PDGFR  PAE/PDGFR  -Alix PDGF-BB (min) 0 30 60 120 0 30 60 120 Ip: PDGFR  Ib: HA-Ub Ip: PDGFR  Ib: PDGFR  Ub-PDGFR  PDGFR  PAE/PDGFR  PAE/PDGFR  -Alix Alix overexpression reduces PDGFR  ubiquitination

12. Alix P P Cbl Ub Alix may stabilize PDGFR  by inducing Cbl degradation

13. Downregulation of receptor tyrosine kinases

14. TC-PTP kowt PDGF-DD - + - + IB: P-Tyr IB: PDGFR  Ligand-activated PDGF  -receptor is hyperphosphorylated in TC-PTP knockout cells relative receptor phosphorylation

15. Decreased rate of PDGF  -receptor degradation in TC-PTP ko MEFs

16. Direct detection of recycled PDGF  -receptors in TC-PTP ko MEFs

17. The PDGF  -receptor co-localizes with Rab4a after 10 min stimulation in TC-PTP ko MEFs PDGF  -receptor Rab4a-EGFP

18. No colocalization between Rab11EGFP and PDGFR  in TC-PTP ko MEFs after 20 min of stimulation

19. degradation TC-PTP? PDGFR  PDGFR  TC-PTP P X P ESCRT? P Y P P X P P P P PTP? Trafficking of PDGF receptors

20. c c c c cc N-glycosylation O-glycosylation Glycosaminoglycan chain c Cysteine residues P PP Phosphorylation site Structure of the hyaluronan receptor CD44 Link domain Hyaluronan-binding domain Basic motif Variant exon (v1-v10) Extracellular Transmembrane Intracellular ERM Actin

21. Co-immunoprecipitation of PDGF ß-receptor and CD44 IP: IB: PDGFR  CD-44 PY99 PDGF-BB: - - + - - + - - + IP: rabbit IgG rabbit IgG rabbit IgG PDGFR  rat IgG CD-44 rat IgG rat IgG IB: CD44 PDGFR  PY99 PDGFR  CD44 PDGFR  CD44

22. Hyaluronan - + + Monoclonal Hermes1, that block hyaluronan binding to CD44, restores PDGF ß- receptor activation Hermes 1 - - + PDGF-BB + + + PY99 PDGFRß Co-exposure of skin fibroblasts to PDGF-BB and hyaluronan inhibits PDGF ß-receptor activation Hyaluronan (µg/ml) 0 10 25 50 100 0 10 25 50 100 PDGF-BB - - - - - + + + + + PY99 PDGFRß

23. PTPases account for the hyaluronan-induced PDGF ß-receptor dephosphorylation Quantification Pervanadate Hyaluronan PDGFR  PDGF-BB PY99 1 1 10 20 100 220 55 225

24. Cross-talk between CD44 and the PDGF ß-receptor CD44  -receptor CD44  -receptor PP P P PTP inactive P P PP PDGF-BB PTP active Signaling Hyaluronan Modulated signaling

25. PDGF in disease Fibrotic conditions lung fibrosis glomerulonephritis liver cirrhosis myelofibrosis Atherosclerosis Malignancies autocrine stimulation (glioblastoma, sarcoma) paracrine stimulation (cancers)

26. Dermatofibro- sarcoma protuberans Chronic mono- myclocytic leukemia Hyperoesinophilic syndrome Gastrointestinal stromal tumor Glioblastoma

27. PDGF antagonists PDGF aptamer -DNA molecule which binds PDGF B-chain with high affinity -specific -expensive, cumbersome to administer STI571 (Glivec, imatinib) -LMW inhibitor of PDGF receptors, c-Kit, Abl, and Arg -not absolutely specific -inexpensive, easy to administer

28. Difference in STI571(Glivec/imatinib) sensitivity between different glioblastoma cultures

29. Clustering of mRNA profiles of glioblastoma cell cultures

30. PDGF 1 3 2 Effects of PDGF in tumors blood vessel stromal fibroblast lymph vessel tumor cells

34. Effect of anti-PDGFR and anti-VEGFR treatment on B16 melanoma tumor growth

35. PDGF and the interstitial fluid pressure (IFP) of tumors PDGF  -receptor activation increases IFP in normal connective tissue Many solid tumors display increased IFP Increased tumor IFP prevents drug uptake into tumors by decreasing transcapillary transport Many solid tumors express PDGF receptors on pericytes and stroma cells

36. Src PLC-  Shc PI3' - kinase SHP-2 Grb2/Sos1 mitogenicity mitogenicity RasMAP kinaseGrb2/Sos1 actin reorganization /chemotaxis /migration Rho, Rac Akt mitogenicity RasMAP kinaseGrb2/Sos1 mitogenicity RasMAP kinase / mitogenicityactin reorganization survival Binding sites for signal transduction molecules on the PDGFR- 

37. 0.5 1 1.5 -1 -2 -3 -4 -0.5 -1.5 -2.5 -3.5 0 10 2030 40 50 60 70 Regulation of Interstitial fluid homeostasis by the PDGFR-  via PI3'kinase mice with wild type PDGFR-  subdermal injection of PDGF-BB injection of C48/80 interstitial fluid pressure pif [mmHg] time [minutes ] mice with PI3´K binding site mutant PDGFR- 

38. Forces that regulate transcapillary transport in tissues b Tumour tissuea Normal tissue Blood vessel Net outward filtration pressure 1-3 mm Hg Net inward or outward pressure (-18-2 mm Hg) COP IF 8 mm Hg P IF -1 to -3 mm Hg COP IF 20 mm Hg P IF 10 -30 mm Hg

39. a Normal tissue b Tumour tissue a Normal tissue b Tumour tissue Structural differences between normal and tumour tissues that affect interstitial fluid pressure

43. PDGF and tumor IFP Tumor-derived PDGF contributes to increased tumor IFP - decreases tumor IFP - synergizes with chemotherapy of tumors Treatment with PDGF antagonist - increases drug uptake in tumors

44. Acknowledgements LICR, UppsalaUppsala University Bengt Westermark Lena Claesson-Welsh Kristofer Rubin Linköping Univ Åke Wasteson Karolinska Institute Monica Nister Arne Östman Christer Betsholtz Lund Univ. in Malmö Lars Rönnstrand Gothenburg Univ. Keiko Funa LICR, Stockholm Ulf Eriksson Johan Lennartsson Piotr Wardega Lotti Rorsman Alexandra Jurek Carina Hellberg Aga Koslowska-Wardega Susann Karlsson Christian Schmees Evi Heldin Lingli Li Rainer Heuchel Monica Krampert Shioto Suzuki Aive Åhgren Ken Amagasaki Gudrun Bäckström Federica Chiara Jean-Baptiste Demoulin Simon Ekman Masao Furuhashi Yuko Hasumi Daniel Hägerstrand Anders Kallin Kaska Kowanetz Ronggui Li Camilla Looman Kristian Pietras Akira Shimizu Tobias Sjöblom