A monoclonal antibody to the type 1 insulin-like growth factor and insulin receptors stimulates deoxyribonucleic acid synthesis in human and murine fibroblasts.
Insulin-like growth factor I (IGF-I) and insulin are polypeptide hormones that stimulate their cellular responses by binding to specific cell membrane receptors. These receptors, while chemically distinct, have similar structural and functional characteristics. This manuscript describes the production and characterization of a monoclonal antibody that binds to both type I IGF and insulin receptors. This antibody did not inhibit hormone binding to either receptor type, but stimulated DNA synthesis in both human and murine fibroblasts. Ten BALB/c-BYJ mice were immunized with human placental membrane fragments, and their splenic lymphocytes were fused with SP2 AG0 mouse myeloma cells. Of approximately 3000 hybridoma clones thus obtained, 1 viable clone, designated V3,8 D7, was found to produce an antibody directed against the type I IGF receptor. Solubilized radiolabeled placental membranes immunoprecipitated with affinity-purified antibody and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed bands with relative molecular masses corresponding to the nonreduced intact receptor (approximately 350 x 10(3], the alpha-subunit (130-140 x 10(3], and the beta-subunit (90 x 10(3] of the type I IGF receptor. Clonal supernatant and affinity-purified antibody precipitated solubilized receptors affinity labeled with [125I]IGF-I. Antibody V3,8 D7 also precipitated solubilized placental membranes affinity labeled with [125I]insulin. However, solubilized receptors affinity purified by the monoclonal antibody bound IGF-I much better than insulin, suggesting that this antibody has a higher affinity for the type I IGF receptor than for the insulin receptor. Affinity-purified antibody did not inhibit the binding of IGF-I or insulin to receptors on human placental membranes, suggesting that it is directed against a site on the type I IGF and insulin receptor not involved in hormone binding. However, affinity-purified monoclonal antibody stimulated DNA synthesis in human GM 498 and murine BALB/c-3T3 clone A 31 fibroblasts, as determined by [3H]thymidine incorporation. The combination of IGF-I and affinity-purified antibody did not increase thymidine incorporation above levels observed with either substrate alone, suggesting that these factors may be operating through a common mechanism. These results suggest that antibody V3,8 D7 can stimulate receptor responses by binding to a site on the type I IGF and/or insulin receptors that is not involved in hormone binding. These data support the concept that hormone receptors themselves possess the biological information required for stimulating specific cellular responses.
Cara
J F
JF
Division of Pediatric Endocrinology and Metabolism, Children's Hospital, Philadelphia, University of Pennsylvania School of Medicine 19104.
Stuart
C A
CA
Furlanetto
R W
RW
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D002478:Cells, Cultured; D004261:DNA Replication; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007334:Insulin-Like Growth Factor I / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010920:Placenta / Q000276:immunology; D011247:Pregnancy; D011972:Receptor, Insulin / Q000276:immunology*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin