Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential.
Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.
Pflanz
S
S
Department of Biochemistry, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany.
Kernebeck
T
T
Giese
B
B
Herrmann
A
A
Pachta-Nick
M
M
Stahl
J
J
Wollmer
A
A
Heinrich
P C
PC
Müller-Newen
G
G
Grötzinger
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D019556:COS Cells; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D050822:Cytokine Receptor gp130; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D018121:Receptors, Cytokine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D003432:Cross-Linking Reagents; C494199:IL6ST protein, human; D008562:Membrane Glycoproteins; D021122:Protein Subunits; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D050822:Cytokine Receptor gp130