Volume 125, Issue 3, Pages 660-667 (September 2003) Gain-of-function mutations of platelet-derived growth factor receptor α gene in gastrointestinal stromal tumors  Seiichi Hirota, Akiko Ohashi, Toshirou Nishida, Koji Isozaki, Kazuo Kinoshita, Yasuhisa Shinomura, Yukihiko Kitamura  Gastroenterology  Volume 125, Issue 3, Pages 660-667 (September 2003) DOI: 10.1016/S0016-5085(03)01046-1

Figure 1 Expression of PDGFR α in GISTs. Northern blotting of representative GISTs was performed with PDGFR α cDNA. Total RNA was loaded, electrophoresed, and transferred to nylon membrane. The membrane was hybridized with 32P-labeled PDGFR α cDNA and autoradiographed. Reprobing with 32P-labeled β actin cDNA was performed. The number corresponds to that of Table 1. Lanes 1–8, GISTs without KIT mutations; lanes 9–15, GISTs with KIT mutations. Gastroenterology 2003 125, 660-667DOI: (10.1016/S0016-5085(03)01046-1)

Figure 2 Mutations of KIT and PDGFR α found in GISTs. (A) Comparison of human KIT and PDGFR α. SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; KI, kinase insert. Arrows indicate 4 different sites in which KIT mutations have been found in GISTs. (B, C) Comparison of the amino acid sequences between human KIT and human PDGFR α at the (B) juxtamembrane domain and (C) TK II domain. (B) PDGFR α mutation at the codon 561-Val to Asp is shown, and (C) PDGFR α mutation at the codon 842-Asp to Val is shown. (C) KIT mutation at codon 816-Asp to Val observed in some human mast cell tumors is shown. Residues that are identical between KIT and PDGFR α are boxed. Amino acids: K, Lys; P, Pro; M, Met; Y, Tyr; E, Glu; V, Val; Q, Gln; W, Trp; I, Ile; N, Asn; G, Gly; D, Asp; T, Thr; L, Leu; H, His; F, Phe; R, Arg; S, Ser; C, Cys; A, Ala. For better alignment, hyphens were introduced in (B). Gastroenterology 2003 125, 660-667DOI: (10.1016/S0016-5085(03)01046-1)

Figure 3 Biochemical and biologic consequences of PDGFR α with the amino acid substitutions. (A) Constitutive tyrosine phosphorylation of mutated PDGFR α. The PDGFR α cDNA with 478-Ser to Pro, 561-Val to Asp, or 842-Asp to Val substitution was transfected into 293T cells. Wild-type PDGFR α was transfected as a control. Tyrosine phosphorylation was examined with or without addition of human PDGF-AA. The membrane was reblotted with anti-PDGFR α antibody. (B) Autonomous proliferation of Ba/F3 cells transfected with mutated PDGFR α cDNA. Mouse type PDGFR α cDNA with 561-Val to Asp or 842-Asp to Val substitution was stably transfected into the IL-3-dependent murine Ba/F3 cells, and tritium thymidine incorporation was determined. Gastroenterology 2003 125, 660-667DOI: (10.1016/S0016-5085(03)01046-1)

Figure 4 Activation of wild-type KIT by binding of constitutively activated PDGFR α. PDGFR α cDNA with 561-Val to Asp or 842-Asp to Val substitution was cotransfected with wild-type KIT cDNA into 293T cells, and immunoprecipitated by anti-KIT polyclonal antibody (A4502). Wild-type PDGFR α cDNA also was cotransfected with wild-type KIT cDNA, and immunoprecipitated with or without human PDGF-AA stimulation. Then tyrosine phosphorylation was examined. As a control, wild-type PDGFR α cDNA was transfected and immunoprecipitated by anti-KIT antibody with or without PDGF-AA stimulation. As another control, wild-type KIT cDNA was transfected and immunoprecipitated by anti-KIT antibody with or without stem cell factor stimulation. The membrane was reblotted with anti-KIT antibody or anti-PDGFR α antibody. Lysate of a GIST tissue with 842-Asp to Val PDGFR α mutation was immunoprecipitated by anti-KIT polyclonal antibody (A4502), and immunoblotting was performed as well. Gastroenterology 2003 125, 660-667DOI: (10.1016/S0016-5085(03)01046-1)

Figure 5 Inhibitory effect of Imatinib mesylate on tyrosine phosphorylation of wild-type and mutated PDGFR α. PDGFR α cDNA with 561-Val to Asp or 842-Asp to Val substitution was transfected into 293T cells, and tyrosine phosphorylation was examined after the treatment with Imatinib mesylate at the concentration of 1 μmol/L or 10 μmol/L. Wild-type PDGFR α cDNA was transfected as a control, and tyrosine phosphorylation was examined after human PDGF-AA stimulation. Gastroenterology 2003 125, 660-667DOI: (10.1016/S0016-5085(03)01046-1)