title: Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.
abstract: Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.
authors.affiliation: Immune Disease Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Li-Zhi
authors.initials: LZ
authors.lastname: Mi
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Grey
authors.forename: Noritaka
authors.initials: N
authors.lastname: Nishida
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
authors.forename: Chafen
authors.initials: C
authors.lastname: Lu
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
mesh_terms: D002460:Cell Line; D004247:DNA / Q000235:genetics; D003902:Detergents; D019281:Dimerization; D004355:Drug Stability; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007668:Kidney / Q000196:embryology; D008823:Micelles; D008854:Microscopy, Electron; D049329:Nanostructures; D010743:Phospholipids / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D003902:Detergents; D008823:Micelles; D010743:Phospholipids; D011994:Recombinant Proteins; D004247:DNA; D066246:ErbB Receptors
references.citation: Immunity. 2006 Oct;25(4):583-94
references.pmid: 17045822
references.citation: Protein Expr Purif. 2001 Dec;23(3):440-6
references.pmid: 11722181
references.citation: Nat Rev Cancer. 2005 May;5(5):341-54
references.pmid: 15864276
references.citation: J Gen Physiol. 2005 Jul;126(1):41-53
references.pmid: 15955874
references.citation: Cell Signal. 2005 Oct;17(10):1183-93
references.pmid: 15982853
references.citation: J Virol. 1988 Oct;62(10):3738-46
references.pmid: 2843671
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11509-14
references.pmid: 16864771
references.citation: Biochemistry. 1987 Mar 10;26(5):1443-51
references.pmid: 3494473
references.citation: J Biol Chem. 1985 Jan 10;260(1):315-9
references.pmid: 2578126
references.citation: Protein Sci. 2003 Nov;12(11):2476-81
references.pmid: 14573860
references.citation: J Biol Chem. 1984 Dec 10;259(23):14631-6
references.pmid: 6094567
references.citation: Biochemistry. 1987 Mar 10;26(5):1434-42
references.pmid: 3494472
references.citation: Annu Rev Pharmacol Toxicol. 2004;44:195-217
references.pmid: 14744244
references.citation: J Biol Chem. 1988 Jan 15;263(2):826-32
references.pmid: 2447082
references.citation: Exp Cell Res. 2003 Mar 10;284(1):78-88
references.pmid: 12648467
references.citation: J Biol Chem. 1980 Nov 25;255(22):10737-43
references.pmid: 6253486
references.citation: Nat Rev Mol Cell Biol. 2001 Feb;2(2):127-37
references.pmid: 11252954
references.citation: Cell. 2006 Jun 16;125(6):1137-49
references.pmid: 16777603
references.citation: Cell. 2002 Sep 20;110(6):775-87
references.pmid: 12297050
references.citation: Science. 2006 May 26;312(5777):1175-8
references.pmid: 16728632
references.citation: Cell. 2002 Sep 20;110(6):763-73
references.pmid: 12297049
references.citation: Science. 2002 Aug 23;297(5585):1330-3
references.pmid: 12154198
references.citation: J Biol Chem. 1992 Dec 25;267(36):26104-9
references.pmid: 1334492
references.citation: Cell. 2000 Oct 13;103(2):211-25
references.pmid: 11057895
references.citation: Cell. 2007 Jul 27;130(2):213-5
references.pmid: 17662934
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19238-43
references.pmid: 18042729
references.citation: Exp Cell Res. 2003 Mar 10;284(1):122-30
references.pmid: 12648471
references.citation: Methods Enzymol. 1991;200:115-20
references.pmid: 1956315
references.citation: Cell. 2006 Jun 16;125(6):1029-31
references.pmid: 16777592
references.citation: Mol Cell. 2003 Feb;11(2):507-17
references.pmid: 12620237
references.citation: J Am Chem Soc. 2004 Mar 24;126(11):3477-87
references.pmid: 15025475
references.citation: J Biol Chem. 1980 May 25;255(10):4834-42
references.pmid: 6246084
references.citation: Arch Biochem Biophys. 2006 Jun 15;450(2):215-22
references.pmid: 16620766
references.citation: Nature. 1984 Oct 4-10;311(5985):480-3
references.pmid: 6090944
references.citation: J Biol Chem. 2002 Nov 29;277(48):46265-72
references.pmid: 12196540
references.citation: Structure. 2006 Apr;14(4):673-81
references.pmid: 16615909
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):190-9
references.pmid: 8742743
references.citation: J Biol Chem. 1982 Feb 10;257(3):1523-31
references.pmid: 6276390
references.citation: J Biol Chem. 1999 Dec 10;274(50):35985-90
references.pmid: 10585488
references.citation: J Biol Chem. 2002 Feb 15;277(7):4704-12
references.pmid: 11741943
references.citation: Biophys J. 2007 Jul 15;93(2):684-98
references.pmid: 17468161
references.citation: Mol Biol Cell. 2002 Jul;13(7):2547-57
references.pmid: 12134089
references.citation: J Biol Chem. 2005 Aug 26;280(34):30392-9
references.pmid: 15994331
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13419-24
references.pmid: 12370423
references.citation: J Biol Chem. 2005 Jun 24;280(25):24043-52
references.pmid: 15840573
references.citation: J Mol Biol. 2005 Apr 8;347(4):759-72
references.pmid: 15769468