Inefficient secretion of human H27A-prolactin, a mutant that does not bind Zn2+.
Human PRL binds Zn2+, but the function of the binding is not known. We investigated the effect on PRL production in pituitary cells by obtaining clones of GH4C1 cells stably transfected with human H27A-PRL, a mutant that does not bind Zn2+. Unexpectedly, clones transfected with the mutant human PRL made little rat PRL. Untransfected GH4C1 cells made between 0.5 to 10 microg rat PRL/10(5) cells in 24 h. Clones transfected with vector alone (four of four), wild type human PRL (six of six), or with human K69A-PRL (two of two) made amounts of rat PRL in the same range. Clones transfected with human H27A-PRL (five of five) made 0.003-0.1 microg rat PRL/10(5) cells in 24 h, and the production of rat PRL mRNA was reduced. Human H27A-PRL was not efficiently secreted; 20-40% newly synthesized H27A-PRL was degraded by 60 min, and there was usually a delay in release of newly synthesized H27A-PRL. Reduction of rat PRL production is not mediated through the PRL receptor, because no sequences for the receptor in GH4C1 cells were detected by RT-PCR. Proteins involved in folding, such as BiP, were not specifically elevated in the H27A-PRL clones. In transient transfections, in which cells have not undergone selection, we found no evidence for disulfide-bonded aggregates of the mutant protein. The results indicate that Zn2+ binding stabilizes PRL in the secretory pathway; the instablility of the mutant protein may trigger effects that suppress rat PRL production directly or that indirectly result in selection of clones with low rat PRL production.
Sun
Z
Z
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Lee
M S
MS
Rhee
H K
HK
Arrandale
J M
JM
Dannies
P S
PS
D000818:Animals; D002460:Cell Line; D006801:Humans; D009154:Mutation; D010902:Pituitary Gland / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
D011388:Prolactin; D015032:Zinc