Cloning and characterization of an alternatively processed human type II interleukin-1 receptor mRNA.
Two types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of approximately 55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1beta as the ligand and shown to have an affinity for human 125I-IL-1beta (KD approximately 2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have approximately 95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5' end identical to the IL-1RII, the 200 base pairs at the 3' end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125I-IL-1beta binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125I-IL-1beta binding (Ki approximately 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced.
Liu
C
C
Neurocrine Biosciences, Inc., San Diego, California 92121, USA.
Hart
R P
RP
Liu
X J
XJ
Clevenger
W
W
Maki
R A
RA
De Souza
E B
EB
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011869:Radioligand Assay; D017472:Receptors, Interleukin-1 / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D017931:DNA Primers; D018076:DNA, Complementary; D007375:Interleukin-1; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1