A ROLE FOR THE CARBOXYL-TERMINUS OF HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR IN THE BINDING OF LIGAND TO THE ALPHA-SUBUNIT OFTHE HIGH-AFFINITY RECEPTOR

A synthetic segment (110-127) of the carboxyl terminus of recombinant human granulocyte-macrophage colony-stimulating factor (rh-GM-CSF) was used to generate a rabbit polyclonal antibody (345-6), which recogniz ed both peptide and full-length Escherichia coli-derived rh-GM-CSF in a direct enzyme-linked immunosorbent assay. Antibody 345-6 was shown t o antagonize the binding of I-125-labeled rh-GM-CSF to its receptor on the KG-1 cell line and to inhibit human GM-CSF-dependent proliferatio n of the AML-193 cell line. The purified IgG fraction of neutralizing antibody 345-6 was used as immunogen to obtain sheep antiserum 1418. A ntibody 1418 recognized antibody 345-6 on direct enzyme-linked immunos orbent assay but did not recognize rh-GM-CSF or the peptide 110-127 to which antibody 345-6 was raised. Antiserum 1418, as well as a purifie d IgG fraction of this serum, inhibited both rh-GM-CSF-stimulated cell proliferation and I-126-labeled rh-GM-CSF receptor binding but not I- 125- labeled recombinant human interleukin-4 receptor binding. The ant i-idiotypic antibody response derived from the anti-(110-127) antibody strongly suggests that the carboxyl-terminal region of rh-GM-CSF may be directly involved in the receptor-ligand interaction of this protei n. The high affinity receptor consists of two different components (GM -R alpha beta) a cytokine-specific alpha-subunit and a beta-subunit th at is shared by human GM-CSF, interleukin-3, and interleukin-5. In an effort to localize the epitope of antibody 1418 to either GMR alpha or GMRB beta several cell lines containing high, low, or both high and l ow affinity receptors were examined. Each was specifically and complet ely inhibited by antibody 1418. Interleukin-3-dependent cell prolifera tion of the AML-193 cell line was found to be unaffected by the antibo dy 1418. Thus, the carboxyl-terminal region of rh-GM-CSF is likely to be involved in the interaction of the ligand with the a-subunit of the high affinity receptor.