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Abstract
A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.
Highlights
A cDNA codingfor the humaninterle~k~nr-e6ceptor tion of cell differentiation, gene activation, and stimulation (IL-6-R) has been expressed stainblmy urine NIH/3T3 or inhib ~ ~ ~ofocnell growth [1, 3]
KDa suggestinga complex o r ~ ~ izati ofnthe IL-6-R in the plasma membraneIL. -6 addedto the transfected NIHf3T3 cells exerted growth inhibition. This antiits response in liver, IL-6 rapidly disappears from this organ and accumulatesin the skin [11].We have presented evidence that Fibroblasts might be responsible for the degradation of IL-6 in skin [11].Because specific degradat~onrequires recognition by surface receptors, we have studied the interaction of recombinant human frh) IL-6 with murine NIW/
Untransfected cells and cells tmsfected with the pBMGNeovectorwithout insert ( l a n e 6 ) do not expressany IL-6-R-mRNA.The extent of overexpressioncan be estimated by comparisonwith the IL-6-R-mRNA expressed by HepG2 cells
Summary
NIH/3T3 cells were transfected stably with the construct pExIRl (Fig. 1) carryingthe entirecodingregionof the human IL-6-R under the transcriptional control of the mouse metallothione~nI promoter. To determinethe dissociationconstant for Iz5I-rhIL-6binding to theIL-6-R, transfected NIH/3T3 cells were incubated with "%rhIL-6 at increasing concentrations. 5 shows control ( A ) and transfected NIH/3T3 cells [23] after incubation with "'I-rhIL-6. A number of 20,000receptors per cell were determined
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