Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high-affinity IL-3 receptor.
Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody-producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL-3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.
Ogorochi
T
T
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104.
Hara
T
T
Wang
H M
HM
Maruyama
K
K
Miyajima
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006825:Hybridomas / Q000276:immunology; D007163:Immunosorbent Techniques; D007377:Interleukin-3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D016185:Receptors, Interleukin-3 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D007377:Interleukin-3; D010446:Peptide Fragments; D016185:Receptors, Interleukin-3; D004247:DNA