Erythropoietin is the prime regulator of red blood cell production. Previous studies demonstrated that antipeptide antibodies to amino acids 99–119 and 111–129 bind to two non-overlapping domains and inhibit the hormone's action (Sytkowski, A. J. & Donahue, K. A. (1987) J. Biol. Chem. 262, 1161–1165). Oligonucleotide-directed mutagenesis now shows that amino acids 99–110 (domain 1) but not 119–129 (domain 2) are important to erythropoietin's structure and function. Mutagenesis of wild-type human erythropoietin cDNA was used to produce a series of mutant proteins with sequential deletion of three adjacent amino acids and insertion of the sequence Glu-Phe across the two domains. Transient expression in COS-7 cells revealed 2.0-kb transcripts encoded by all of the cDNAs. Domain 2 mutants exhibited specific biological activities similar to that of the wild type. In contrast, domain 1 mutants were not secreted. In vitro transcription and translation of the domain 1, domain 2 and wild-type cDNAs resulted in the isolation of 23.5-kDa and 32-kDa proteins in the absence or presence of pancreatic microsomes, respectively, consistent with efficient translation of all of the mutants and equivalent post-translational processing of each protein. The data suggest that mutation within domain 1 results in the intracellular biosynthesis of erythropoietins with altered structure, rendering them subject to rapid degradation. The bioassay of erythropoietins synthesized entirely in vitro demonstrated that domain 1 mutants were inactive, whereas both wild type and domain 2 mutant hormones exhibited biologic activity. The results are consistent with a critical role for amino acids 99–110 in the structure of human erythropoietin.
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erythropoietin cDNA insert