title: Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase.
abstract: The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.
authors.affiliation: From the Department of Structural Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of RAS, 117997 Moscow, Russia, the Faculty of Medicine, Moscow State University, 119992 Moscow, Russia.
authors.forename: George V
authors.initials: GV
authors.lastname: Sharonov
authors.affiliation: From the Department of Structural Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of RAS, 117997 Moscow, Russia.
authors.forename: Eduard V
authors.initials: EV
authors.lastname: Bocharov
authors.affiliation: the Department of Molecular Neurobiology, Institute of Higher Nervous Activity and Neurophysiology of RAS, 117485 Moscow, Russia, and.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kolosov
authors.affiliation: From the Department of Structural Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of RAS, 117997 Moscow, Russia.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Astapova
authors.affiliation: From the Department of Structural Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of RAS, 117997 Moscow, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Arseniev
authors.affiliation: From the Department of Structural Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of RAS, 117997 Moscow, Russia, the Biological Faculty, Moscow State University, 119992 Moscow, Russia avfeofanov@yandex.ru.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Feofanov
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D017354:Point Mutation; D055503:Protein Multimerization / Q000235:genetics*; D017434:Protein Structure, Tertiary; D036104:Receptor, EphA2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D036104:Receptor, EphA2
keywords: Alternative Dimerization; Cell Surface Receptor; Eph Receptor; Flow Cytometry; Membrane Proteins; Protein Domains; Receptor Tyrosine Kinase; Transmembrane Domain
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):16-25
references.pmid: 22040915
references.citation: Mol Cells. 2010 May;29(5):443-8
references.pmid: 20432069
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13658-63
references.pmid: 16954199
references.citation: Int Immunol. 1998 Jul;10(7):923-33
references.pmid: 9701030
references.citation: Biochemistry. 2008 Nov 11;47(45):11771-82
references.pmid: 18855427
references.citation: Biochim Biophys Acta. 2008 Oct;1778(10):2361-7
references.pmid: 18590698
references.citation: J Cell Biol. 2004 Mar 1;164(5):661-6
references.pmid: 14993233
references.citation: Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14278-83
references.pmid: 16179394
references.citation: Hum Reprod. 2011 May;26(5):1163-70
references.pmid: 21349856
references.citation: Proteins. 2013 Mar;81(3):365-76
references.pmid: 23042146
references.citation: Nat Struct Mol Biol. 2010 Apr;17(4):398-402
references.pmid: 20228801
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15937-40
references.pmid: 12461170
references.citation: Biochemistry (Mosc). 2012 Dec;77(12):1387-94
references.pmid: 23244735
references.citation: J Am Chem Soc. 2013 Jun 5;135(22):8105-8
references.pmid: 23679838
references.citation: Science. 1992 Oct 23;258(5082):659-62
references.pmid: 1329208
references.citation: Oncogene. 2014 May 15;33(20):2568-76
references.pmid: 23752188
references.citation: J Cell Sci. 2008 Oct 1;121(Pt 19):3207-17
references.pmid: 18782861
references.citation: Biophys J. 2010 Mar 3;98(5):881-9
references.pmid: 20197042
references.citation: Circ Res. 2011 Apr 29;108(9):1071-83
references.pmid: 21415392
references.citation: Structure. 2008 Jul;16(7):991-1001
references.pmid: 18611372
references.citation: J Biol Chem. 2008 Oct 24;283(43):29385-95
references.pmid: 18728013
references.citation: J Biol Chem. 2008 Mar 21;283(12):7733-44
references.pmid: 18201969
references.citation: Cell. 2010 Jun 25;141(7):1117-34
references.pmid: 20602996
references.citation: Cell Adh Migr. 2010 Apr-Jun;4(2):284-98
references.pmid: 20421711
references.citation: Oncogene. 2011 Dec 15;30(50):4921-9
references.pmid: 21666714
references.citation: Phys Biol. 2010 Mar 12;7:16014
references.pmid: 20228445
references.citation: Biochemistry. 2014 Jan 21;53(2):323-32
references.pmid: 24397339
references.citation: J Neurosci. 2003 Dec 17;23(37):11523-38
references.pmid: 14684856
references.citation: Oncogene. 2001 Jun 14;20(27):3553-62
references.pmid: 11429702
references.citation: Structure. 2013 Nov 5;21(11):2087-93
references.pmid: 24120763
references.citation: EMBO J. 1992 Mar;11(3):923-32
references.pmid: 1347745
references.citation: Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10860-5
references.pmid: 20505120
references.citation: J Cell Sci. 1999 Dec;112 ( Pt 23):4415-23
references.pmid: 10564659
references.citation: Am J Pathol. 2009 Jun;174(6):2388-99
references.pmid: 19443703
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1424-9
references.pmid: 15671157
references.citation: Mol Biol Cell. 2008 Feb;19(2):646-54
references.pmid: 18045991
references.citation: Oncogene. 1997 Mar 27;14(12):1397-406
references.pmid: 9136983
references.citation: Biochim Biophys Acta. 2012 Apr;1818(4):963-73
references.pmid: 21827736
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):43-50
references.pmid: 22261642
references.citation: J Biol Chem. 2008 Jun 6;283(23):16017-26
references.pmid: 18387945
references.citation: Expert Opin Ther Targets. 2011 Jan;15(1):31-51
references.pmid: 21142802
references.citation: Biotechniques. 1990 Apr;8(4):404-7
references.pmid: 2340178
references.citation: Bioinformatics. 2014 Mar 15;30(6):889-90
references.pmid: 24202542
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):92-101
references.pmid: 22040910
references.citation: J Biol Chem. 2012 Dec 21;287(52):43810-24
references.pmid: 23105112
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):35-42
references.pmid: 22044883
references.citation: Cell Adh Migr. 2010 Apr-Jun;4(2):313-24
references.pmid: 20543559
references.citation: Cell Signal. 2012 Aug;24(8):1522-30
references.pmid: 22504297
references.citation: EMBO Rep. 2009 Jul;10(7):722-8
references.pmid: 19525919
references.citation: Biochim Biophys Acta. 2012 Feb;1818(2):183-93
references.pmid: 21910966