Conversion of bovine growth hormone cysteine residues to serine affects secretion by cultured cells and growth rates in transgenic mice.
GHs have been found to possess two disulfide bonds. We set out to determine the importance of bovine (b) GH's disulfide bonds relative to the ability of the hormone to be secreted by cultured cells in vitro and to promote growth in transgenic mice. We have generated six mutated bGH genes that encode serine (Ser) substitutions for cysteines (Cys). These mutated genes were used to generate bGH analogs in which either one or both disulfide bonds are destroyed. When the small loop of bGH was destroyed (Cys181-Ser or Cys189-Ser), the bGH analogs were found to be secreted by mouse L-cells at levels comparable to those of wild-type bGH. However, secretion was drastically reduced when the large loop was abolished (Cys53-Ser or Cys164-Ser). An immunofluorescence study of these bGH analogs revealed two distinct patterns of subcellular localization. Bovine GH analogs with mutations in the small loop demonstrated a perinuclear distribution similar to that of wild-type bGH, but analogs containing a disrupted large loop revealed a uniform cytoplasmic distribution pattern. When these mutated bGH genes were individually introduced into transgenic mice, only those animals that expressed bGH analogs with the large loop intact demonstrated a growth-enhanced phenotype. Transgenic mice that expressed bGH analogs lacking the large loop showed growth rates similar to those of nontransgenic mice. These results suggest that the integrity of the large loop, but not that of the small loop, is essential for the growth-enhancing activity of bGH in transgenic mice.
Chen
X Z
XZ
Molecular and Cellular Biology Program, Edison Animal Biotechnology Center, Ohio University, Athens 45701.
Shafer
A W
AW
Yun
J S
JS
Li
Y S
YS
Wagner
T E
TE
Kopchick
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001835:Body Weight; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine; D004220:Disulfides; D005455:Fluorescent Antibody Technique; D006128:Growth / Q000502:physiology*; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007739:L Cells; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D011986:Receptors, Somatotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine
D004220:Disulfides; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D012694:Serine; D013006:Growth Hormone; D003545:Cysteine