FUNCTIONAL AND STRUCTURAL ROLE OF ARGININE-103 IN HUMAN ERYTHROPOIETIN

The identification of amino acid residues essential for function of th e hematopoietic growth factor erythropoietin has been approached by se veral methods, including comparisons of related sequences, immunochemi cal approaches, mutagenesis, and computer modeling. We have reported p reviously that mutations within amino acids 100-109 of erythropoietin can have profound effects on the hormone's structure and/or activity a nd that Arg103 is especially important for function [Y. Chern, T. Chun g, and A. J. Sytkowski (1991) Eur. J. Biochem. 202, 225-229; J. Grodbe rg, K. L. Davis, and A. J. Sytkowski (1993) Eur. J. Biochem. 218, 597- 601]. We have now constructed a series of Arg103 substitutions in orde r to determine the structural features of amino acid 103 required for biological activity, Each of the mutants was expressed and secreted ef ficiently by transfected COS1 cells. Mutants Arg103Asn, Arg103Gln, and Arg103Glu exhibited no biological activity. In contrast, Arg103His an d Arg103Lys had specific activities equal to 2 and 25%, respectively, of that of wild-type erythropoietin, indicating that a positive charge may be required at position 103 but that other constraints necessitat e the presence of Arg for full activity. A role for amino acid 103 in the protein's structure was supported by the results of experiments wh ich revealed marked differences in heat stability among the mutants. W e hypothesize that an Arg at position 103 may confer sufficient flexib ility to the receptor binding domain to facilitate initial binding to the receptor and may then stabilize the binary complex by hydrogen bon ding with carbonyls of the receptor protein. (C) 1996 Academic Press, Inc.