title: A single amino acid substitution converts a transmembrane protein activator of the platelet-derived growth factor β receptor into an inhibitor.
abstract: Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFβ receptor (PDGFβR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFβR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFβR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFβR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFβR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFβR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFβR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFβR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFβR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.
authors.affiliation: Department of Genetics, Department of Molecular Biophysics and Biochemistry, Department of Therapeutic Radiology, and the Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Petti
authors.affiliation: Yale University School of Public Health, New Haven, Connecticut 06520.
authors.forename: Kristina
authors.initials: K
authors.lastname: Talbert-Slagle
authors.affiliation: University of California at Berkeley, Berkeley, California 94704.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Hochstrasser
authors.affiliation: Department of Genetics, Department of Molecular Biophysics and Biochemistry, Department of Therapeutic Radiology, and the Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510. Electronic address: Daniel.dimaio@yale.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: DiMaio
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008297:Male; D051379:Mice; D020125:Mutation, Missense; D020575:Oncogene Proteins v-sis / Q000235:genetics / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000378:metabolism*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D020536:Enzyme Activators; D020575:Oncogene Proteins v-sis; D009856:Oncogene Proteins, Viral; D047428:Protein Kinase Inhibitors; C054304:oncogene protein E5, Bovine papillomavirus type 1; D020797:Receptor, Platelet-Derived Growth Factor beta
keywords: Cell Signaling; E5 Protein; PDGF Receptor; Protein Engineering; Protein-Protein Interactions; Receptor Tyrosine Kinase; Site-directed Mutagenesis; Transformation; Traptamers; Tumor Viruses
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15937-40
references.pmid: 12461170
references.citation: Proteins. 1998 Dec 1;33(4):601-12
references.pmid: 9849943
references.citation: Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15241-6
references.pmid: 9860953
references.citation: Cell. 1989 Sep 22;58(6):1121-33
references.pmid: 2550144
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 29;110(5):1646-51
references.pmid: 23319611
references.citation: Cell. 2013 Jan 31;152(3):543-56
references.pmid: 23374349
references.citation: J Virol. 1999 Apr;73(4):3264-72
references.pmid: 10074180
references.citation: Mol Cell Biol. 1988 Oct;8(10):4071-8
references.pmid: 2847028
references.citation: Nat Cell Biol. 2001 May;3(5):517-21
references.pmid: 11331882
references.citation: Biochim Biophys Acta. 1998 Aug 19;1378(1):F79-113
references.pmid: 9739761
references.citation: J Virol. 1995 Sep;69(9):5869-74
references.pmid: 7543592
references.citation: Nat Cell Biol. 2001 May;3(5):512-6
references.pmid: 11331881
references.citation: Cancer Gene Ther. 2003 Jan;10(1):57-63
references.pmid: 12489029
references.citation: Adv Cancer Res. 2007;97:247-74
references.pmid: 17419949
references.citation: J Virol. 1997 Oct;71(10):7318-27
references.pmid: 9311809
references.citation: J Biol Chem. 2002 Dec 6;277(49):47149-59
references.pmid: 12351659
references.citation: J Virol. 1998 Nov;72(11):8921-32
references.pmid: 9765437
references.citation: Mol Cell Biol. 1993 Jul;13(7):4137-45
references.pmid: 8321218
references.citation: Science. 1983 Jul 15;221(4607):275-7
references.pmid: 6304883
references.citation: EMBO J. 1991 Apr;10(4):845-55
references.pmid: 1849073
references.citation: J Virol. 2002 Aug;76(16):7976-86
references.pmid: 12134002
references.citation: J Mol Biol. 2001 Aug 31;311(5):1011-26
references.pmid: 11531336
references.citation: J Virol. 1995 Oct;69(10):6507-17
references.pmid: 7666552
references.citation: Cytokine Growth Factor Rev. 2004 Aug;15(4):197-204
references.pmid: 15207811
references.citation: J Mol Biol. 2004 May 14;338(5):907-20
references.pmid: 15111056
references.citation: Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6939-43
references.pmid: 7688466
references.citation: Mol Cell Biol. 1995 May;15(5):2570-81
references.pmid: 7739538
references.citation: EMBO J. 1987 Aug;6(8):2381-5
references.pmid: 2822390
references.citation: Cell. 1995 Jan 27;80(2):213-23
references.pmid: 7834741
references.citation: J Biol Chem. 1993 Jun 5;268(16):11500-3
references.pmid: 8389353
references.citation: Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6736-40
references.pmid: 1323117
references.citation: Cell Growth Differ. 2000 Jul;11(7):395-408
references.pmid: 10939593
references.citation: Biochemistry. 2008 Aug 26;47(34):8985-92
references.pmid: 18672907
references.citation: Proc Natl Acad Sci U S A. 1994 May 24;91(11):4634-8
references.pmid: 8197111
references.citation: J Mol Biol. 2005 Jan 28;345(4):907-21
references.pmid: 15588835
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):11945-50
references.pmid: 17609376
references.citation: J Virol. 2012 Mar;86(6):3014-26
references.pmid: 22238299
references.citation: Science. 1986 Jul 25;233(4762):464-7
references.pmid: 3014660
references.citation: Mol Cell Biol. 1991 Feb;11(2):1125-32
references.pmid: 1703628
references.citation: Mol Biol Cell. 2000 Oct;11(10):3589-99
references.pmid: 11029057
references.citation: J Virol. 2009 Oct;83(19):9773-85
references.pmid: 19605488
references.citation: J Biol Chem. 1999 Apr 9;274(15):10249-58
references.pmid: 10187811
references.citation: Oncogene. 1995 Apr 6;10(7):1431-9
references.pmid: 7731695
references.citation: Cell. 2013 Jan 31;152(3):557-69
references.pmid: 23374350
references.citation: Nature. 1983 Jul 7-13;304(5921):35-9
references.pmid: 6306471
references.citation: J Biol Chem. 1993 Oct 15;268(29):21478-81
references.pmid: 7691811
references.citation: Biophys J. 2010 Sep 22;99(6):1764-72
references.pmid: 20858420
references.citation: EMBO J. 1992 Dec;11(13):4851-9
references.pmid: 1334459