Mutational analysis of evolutionarily conserved ACTH residues.
alpha-Melanocyte stimulating hormone (MSH) and adrenocorticotropin (ACTH)1-24, the minimal ACTH sequence required for full activity, differ only by the 10 C-terminal amino acids of ACTH1-24. Interestingly, these ten C-terminal residues have been highly conserved throughout vertebrate evolution. To understand the functional constraints of these 10 amino acids we analyzed the effects of mutating these residues on steroidogenic activity in vivo and in vitro. Alanine substitutions of some of the first four amino acid residues (the basic core residues KKRR, 15-18) greatly reduces ACTH activity in vitro and in vivo; replacement of mutant alanines at residues 15 and 17 with glutamine residues partially restores ACTH activity. Thus, for ACTH receptor binding and activation, the amino acid residues 15-18 are important for their side chains. Surprisingly, conversion of the five C-terminal residues (20-24) to alanines increases ACTH activity in vivo over that of native ACTH. With respect to receptor binding and activity, the last five amino acid residues are important only for the peptide length they contribute; however, with respect to serum stability, their side chains are significant.
Developmental Biology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA. jcosta@du.edu
Jessica L
JL
Costa
Stephanie
S
Bui
Peggy
P
Reed
Robert M
RM
Dores
Miles B
MB
Brennan
Ute
U
Hochgeschwender
D000311:Adrenal Glands / Q000378:metabolism; D000324:Adrenocorticotropic Hormone / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D002417:Cattle; D017124:Conserved Sequence; D053497:Corticotropin-Like Intermediate Lobe Peptide; D004252:DNA Mutational Analysis; D005399:Fishes; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D009074:Melanocyte-Stimulating Hormones / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D014981:Xenopus
D010446:Peptide Fragments; D053497:Corticotropin-Like Intermediate Lobe Peptide; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D009074:Melanocyte-Stimulating Hormones