Dynamics of the gp130 cytokine complex: a model for assembly on the cellular membrane.
Cytokines of the interleukin-6 (IL-6)-type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL-6, interleukin-11, ciliary neurotrophic factor, neuropoietin, cardiotrophin-1, and cardiotrophin-1-like-cytokine, interact with specific ligand binding receptor proteins. High- and low-affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine-receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc-fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL-6 and interleukin-b receptor (IL-6R) by means of fluorescence-correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell-free condition the binding affinities and dynamics of IL-6 and IL-6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL-6/IL-6R complex and only at higher ligand concentrations does it bind a second IL-6/IL-6R complex. This view contrasts with the current perception of IL-6 receptor activation and reveals an alternative receptor activation mechanism.
Schroers
Andreas
A
Evotec Technologies GmbH, D-40225 Düsseldorf, Germany.
Hecht
Oliver
O
Kallen
Karl-Josef
KJ
Pachta
Michael
M
Rose-John
Stefan
S
Grötzinger
Joachim
J
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D050822:Cytokine Receptor gp130; D003627:Data Interpretation, Statistical; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D015703:Antigens, CD; D007141:Immunoglobulin Fc Fragments; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130
Biophys J. 2000 Dec;79(6):2858-66
11106594
Stem Cells. 2002;20(2):119-38
11897869
J Biol Chem. 1999 Apr 23;274(17):11859-67
10207005
J Mol Biol. 1994 Sep 30;242(4):497-507
7932706
Blood. 2003 Aug 15;102(4):1186-95
12714508
Science. 2003 Jun 27;300(5628):2101-4
12829785
Biol Chem. 1999 Jul-Aug;380(7-8):803-13
10494829
J Exp Med. 1996 Apr 1;183(4):1399-406
8666898
Trends Biotechnol. 2002 Oct;20(10):417-9
12220903
Eur Cytokine Netw. 2000 Mar;11(1):27-38
10705296
Mol Cell. 2003 Sep;12(3):577-89
14527405
Neuron. 1996 Jul;17(1):63-74
8755479
Biophys J. 1976 Nov;16(11):1315-29
974223
Blood. 1998 Nov 15;92(10):3495-504
9808540
J Exp Med. 1997 Feb 17;185(4):755-66
9034153
Eur J Biochem. 2001 Jan;268(1):160-7
11121117
EMBO J. 1995 May 1;14(9):1942-51
7744001
Mol Cell Proteomics. 2002 Oct;1(10):805-15
12438563
Annu Rev Immunol. 1997;15:797-819
9143707
Eur J Neurosci. 1999 Sep;11(9):2995-3004
10510164
Nat Cell Biol. 2000 Mar;2(3):168-72
10707088
Science. 2001 Mar 16;291(5511):2150-5
11251120
J Biol Chem. 1996 Dec 20;271(51):32635-43
8999038
EMBO J. 1996 Jun 3;15(11):2726-37
8654370
J Biol Chem. 1997 Jun 20;272(25):15760-4
9188471
Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4827-32
15051883
Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3251-6
9501249
J Biol Chem. 1994 Sep 16;269(37):23286-9
8083235
J Immunol. 1992 Sep 15;149(6):2021-7
1381393
J Immunol. 1999 Oct 15;163(8):4583-9
10510402
Science. 1988 Aug 12;241(4867):825-8
3136546
FEBS Lett. 1992 Jul 20;306(2-3):219-22
1321736
Science. 1999 Feb 12;283(5404):987-90
9974392
Cell. 1989 Aug 11;58(3):573-81
2788034
Biochem J. 1994 Jun 1;300 ( Pt 2):281-90
8002928
J Biomol Screen. 2001 Feb;6(1):29-37
11679163
J Immunol. 2000 Dec 15;165(12):7042-9
11120832
Adv Exp Med Biol. 2001;495:145-51
11774558
Biochim Biophys Acta. 2002 Nov 11;1592(3):251-63
12421670
Nat Biotechnol. 1997 Feb;15(2):142-5
9035138
Anal Biochem. 1993 May 1;210(2):425-8
8512082
Drug Discov Today. 2001 Jun 1;6(12):637-646
11408200
J Immunol. 1999 Feb 1;162(3):1480-7
9973404
Cytokine. 1992 Mar;4(2):96-100
1633265
Eur J Biochem. 1995 Dec 1;234(2):661-9
8536717
J Biol Chem. 1996 Aug 16;271(33):20138-44
8702737