Inefficient secretion of human H27A-prolactin, a mutant that does not bind Zn2+
| Autor: | Joanne M. Arrandale, Min S. Lee, Harrison K. Rhee, Priscilla S. Dannies, Zhenyu Sun |
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| Rok vydání: | 1997 |
| Předmět: |
endocrine system
endocrine system diseases Mutant Biology Transfection Polymerase Chain Reaction Cell Line Endocrinology Mutant protein Animals Humans Secretion Receptor Molecular Biology Messenger RNA Wild type General Medicine Molecular biology Prolactin Rats Zinc Cell culture Pituitary Gland Mutation hormones hormone substitutes and hormone antagonists Protein Binding Signal Transduction |
| Zdroj: | Molecular endocrinology (Baltimore, Md.). 11(10) |
| ISSN: | 0888-8809 |
| Popis: | 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 μg rat PRL/105 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 μg rat PRL/105 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. |
| Databáze: | OpenAIRE |
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