In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130.
Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha-receptor (Ralpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6Ralpha/gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its Ralpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-Ralpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6Ralpha, and 1 gp130-CHR. Addition of the N-terminal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6Ralpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.
Chow
D
D
Departments of Microbiology & Immunology, and Structural Biology, Stanford University School of Medicine, Fairchild D319, 299 Campus Drive, Stanford, California 94305-5124, USA.
Ho
J
J
Nguyen Pham
T L
TL
Rose-John
S
S
Garcia
K C
KC
D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050822:Cytokine Receptor gp130; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D019947:Receptors, Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
D015703:Antigens, CD; D016207:Cytokines; C494199:IL6ST protein, human; D007136:Immunoglobulins; D015850:Interleukin-6; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D050822:Cytokine Receptor gp130