Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis.
Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10(5)-10(6) receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.
Nagy
Peter
P
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Claus
Jeroen
J
Jovin
Thomas M
TM
Arndt-Jovin
Donna J
DJ
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein
Biophys J. 1993 Nov;65(5):2021-40
8298032
J Cell Biol. 1995 Jun;129(6):1543-58
7790353
Biophys J. 2007 Aug 1;93(3):1021-31
17496034
J Cell Biol. 2005 Aug 15;170(4):619-26
16103229
Cytometry. 1999 Apr 1;35(4):353-62
10213201
Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2170-5
19168634
Biophys J. 2007 Jul 15;93(2):684-98
17468161
J Biol Chem. 2005 Aug 26;280(34):30392-9
15994331
Mol Cell. 2003 Feb;11(2):495-505
12620236
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4995-5000
10220407
Curr Opin Cell Biol. 2009 Apr;21(2):177-84
19208461
Cell. 2009 Jun 26;137(7):1293-307
19563760
Exp Cell Res. 2009 Feb 15;315(4):683-96
19278030
Eur Biophys J. 2008 Apr;37(4):469-81
18043914
Biochim Biophys Acta. 2005 Dec 30;1746(3):234-51
16368465
Exp Cell Res. 2009 Feb 15;315(4):638-48
19038249
J Gen Physiol. 2005 Jul;126(1):41-53
15955874
Biophys J. 2008 Aug;95(3):1349-59
18359791
Biophys J. 2008 Mar 15;94(6):2320-32
18096627
Nature. 2003 Feb 13;421(6924):756-60
12610629
Cell. 2006 Jun 16;125(6):1137-49
16777603
Nature. 2010 Apr 1;464(7289):783-7
20208517
Cell. 2002 Sep 20;110(6):775-87
12297050
Biophys J. 2008 Aug;95(4):2086-96
18487307
Biophys J. 2007 May 1;92(9):3098-104
17416632
Cytometry A. 2009 Aug;75(8):650-7
19526485
Nat Biotechnol. 2004 Feb;22(2):198-203
14704683
Biochemistry. 2007 Dec 18;46(50):14403-17
18027983
J Cell Sci. 2002 Nov 15;115(Pt 22):4251-62
12376557
Exp Cell Res. 2009 Feb 15;315(4):649-58
18761339
J Biol Chem. 2004 Aug 27;279(35):36972-81
15215236
Cancer Cell. 2004 Apr;5(4):317-28
15093539
Biochemistry. 2007 Apr 17;46(15):4589-97
17381163
ACS Chem Biol. 2008 May 16;3(5):287-93
18484709
J Biol Chem. 2001 May 4;276(18):15256-63
11278603
Proc Natl Acad Sci U S A. 1981 Nov;78(11):6981-5
6273899
Biochem Soc Trans. 2009 Oct;37(Pt 5):955-60
19754431
J Biol Chem. 1988 Mar 5;263(7):3290-5
3257758
Cytometry. 1998 Jun 1;32(2):120-31
9627225
Nat Cell Biol. 2008 Aug;10(8):955-63
18641640
J Cell Physiol. 2010 Sep;224(3):649-57
20432461
Growth Factors. 2008 Dec;26(6):316-24
18937111
Biochem Soc Trans. 2003 Oct;31(Pt 5):1020-7
14505472
J Am Chem Soc. 2005 Sep 21;127(37):12770-1
16159249
Biochemistry. 1987 Mar 10;26(5):1443-51
3494473
J Phys Chem B. 2009 Dec 31;113(52):16654-9
19928819
Biophys J. 2002 May;82(5):2415-27
11964230
Biochemistry. 2007 Feb 20;46(7):2010-9
17253768
J Cell Sci. 1999 Jun;112 ( Pt 11):1733-41
10318765
J Biol Chem. 2002 Aug 9;277(32):28468-73
12000754
Sci STKE. 2002 Nov 26;2002(160):re15
12454318
Biophys J. 2009 Nov 4;97(9):2613-22
19883605
J Biol Chem. 1999 Mar 26;274(13):8865-74
10085130
Cancer Cell. 2008 Apr;13(4):365-73
18394559
J Biol Chem. 1999 Oct 22;274(43):30636-43
10521449