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Inefficient Secretion of Human H27A-Prolactin, a Mutant That Does Not Bind Zn²⁺

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

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.

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