Functional analysis of the cysteine residues of activin A.
Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor. Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers. A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues. Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold). Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells. Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A. Cys 113 monomers had undetectable levels of biological activity. No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115. The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity. Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Endocrine Research, Genentech Inc., South San Francisco, California 94080.
A J
AJ
Mason
D028341:Activins; D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000032:analysis* / Q000502:physiology*; D004926:Escherichia coli; D006801:Humans; D007265:Inhibins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000032:analysis / Q000502:physiology; D014162:Transfection
D011498:Protein Precursors; D028341:Activins; D007265:Inhibins; D003545:Cysteine; D000409:Alanine