Molecular cloning, characterization and functional expression of the rat liver interleukin 6 receptor.
A series of cDNA clones coding for the rat liver interleukin 6 receptor (IL6-R) were isolated from an acute-phase library. The identity of the clones was established (a) by DNA sequence analysis and comparison with the known human leukocyte IL6-R and (b) by demonstrating that the clones generated specific IL6 ligand binding activity and IL6-dependent regulation of acute-phase gene control elements after transfection into appropriate recipient cells. Two types of cDNA clones were obtained corresponding to two mRNA species of different length, both encoding an identical protein of 462 amino acid residues. The two prototype clones, pRIL6RC.21 and pRIL6RC.6 contained 3'-untranslated regions of 550 and 3100 nucleotides, respectively. The sequence motifs TTATTTAT and ATTTA associated with the regulation of mRNA stability and translation efficiency were present only in the longer mRNA species. The deduced amino acid sequence of the rat liver IL6-R was 53% identical with the human leukocyte IL6-R. Both receptors contained conserved structural features in their extracellular domains, including the signal peptide, a C2 domain characteristic of the immunoglobulin superfamily, and two domains shared among members of a family of cytokine and growth factor receptors. The strongly conserved intracellular portion of the rat liver IL6-R lacked recognizable signal transduction domains. The cDNA clones were used to demonstrate that rat liver IL6-R mRNA concentrations were increased 4.2-fold at 12 h after the induction of an experimental acute-phase response. Clone pRIL6R.21ex, but not clone pRIL6RC.6ex, generated specific IL6 ligand binding activity after transfection into human Jurkat cells that lack the endogenous IL6-R. By contrast, only pRIL6RC.6ex reconstituted a response of human Hep3B-2 and HepG2 hepatoma cells to mouse IL6. These human hepatoma cells were highly responsive to human IL6 but did not respond to physiologic concentrations of murine IL6. After cotransfection with pRIL6RC.6ex and plasmids containing the chloramphenicol acetyltransferase reporter gene under the control of IL6 response elements of acute-phase plasma protein genes, these cells showed a strong stimulation of the reporter gene by recombinant mouse IL6. Thus, both cell surface ligand binding activity and the complete IL6 signal cascade terminating in the transcriptional induction of IL6-dependent promoters were successfully reconstituted. Therefore, both IL6-R mRNA species code for functionally active receptor, depending on the target cell, but only the longer mRNA species coded for significant receptor levels in human hepatoma cell lines.
Baumann
M
M
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Baumann
H
H
Fey
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism / Q000494:pharmacology; D007962:Leukocytes / Q000737:chemistry; D008099:Liver / Q000737:chemistry*; D008113:Liver Neoplasms; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011916:Rats, Inbred F344; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D019947:Receptors, Interleukin-6; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D012333:RNA, Messenger; D011971:Receptors, Immunologic; D019947:Receptors, Interleukin-6; D004247:DNA