Effects of a leucine analog on growth hormone processing and secretion by cultured cells.
Bovine and rat growth hormones (bGH and rGH, respectively) possess signal peptides that direct the hormone to the secretory pathway and are proteolytically cleaved prior to secretion. Previous in vitro translation studies indicated that incorporation of the polar leucine analog beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal peptide function. To test the effects of this analog on GH secretion by cultured animal cells, transfections of mouse L-cells with a bGH expression plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells was performed in the absence or presence of beta-hydroxyleucine. Transient expression of bGH in mouse L-cells or endogenous expression of rGH in anterior pituitary cells resulted in an accumulation of GH in the culture medium. Treatment with beta-hydroxyleucine resulted in a block in secretion as evidenced by an accumulation of GHs within these cells. Amino-terminal sequencing of the intracellular form of the analog-substituted GHs demonstrated accurate signal peptide cleavage. In contrast, in vitro translations of bGH RNA performed in the presence of beta-hydroxyleucine and microsomal membranes resulted in the inhibition of signal peptide cleavage. The results suggest that beta-hydroxyleucine can uncouple signal peptide processing and protein secretion in cultured cells.
McAndrew
S J
SJ
Department of Zoology, Ohio University, Athens 45701.
Chen
N Y
NY
Kelder
B
B
Cioffi
J A
JA
Kopchick
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000378:metabolism*; D006868:Hydrolysis; D007739:L Cells; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection
D021382:Protein Sorting Signals; C024584:beta-hydroxyleucine; D013006:Growth Hormone; D007930:Leucine