Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.
Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.
Delafontaine
P
P
Department of Medicine, Emory University, Atlanta, GA 30322.
Ku
L
L
Ververis
J J
JJ
Cohen
C
C
Runge
M S
MS
Alexander
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D002478:Cells, Cultured; D018604:Epitope Mapping; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D010455:Peptides / Q000276:immunology*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000276:immunology*; D012995:Solubility
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010455:Peptides; D017526:Receptor, IGF Type 1