A Molecular Mimic of Phosphorylated Prolactin (S179D PRL) Secreted by Eukaryotic Cells Has a Conformation with an Increased Positive Surface Charge Compared to That of Unmodified Prolactin

摘要

S179D prolactin (S179D PRL) is a pseudophosphorylated form of human PRL which has potentnantitumor and anti-angiogenic activities in vivo. This molecule binds to the same forms of the PRL receptorn(PRLR) as unmodified PRL, yet this binding results in different intracellular signaling and biological endnpoints. Since it is now clear that PRLRs are predimerized and therefore that ligand binding must initiatensignaling by inducing a conformational change in the receptor dimer, we hypothesized that S179D PRL hadnan altered conformation compared to unmodified PRL. The conformation of the ligand-receptor ternaryncomplex would therefore also have an altered conformation, and thus, different signaling molecules would benactivated. Here we present evidence in support of this hypothesis by demonstrating, in contrast to unmodifiednPRL, that S179D PRL has reduced nickel and zinc binding capacity and a higher affinity for heparin andnDEAE. Conformational changes have occurred since these features are counterintuitive on the basis of thensimple substitution of a serine with a negatively charged aspartate residue. To demonstrate that thesenparticular properties of S179D PRL were not due to misfolding of the molecule during production, S179DnPRL was expressed in two different mammalian cell lines. Also investigated was the potential for productionnof S179D PRL as a soluble cytoplasmic, or secreted periplasmic, protein in Escherichia coli.