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.
S179D prolactin (S179D PRL) is a pseudophosphorylated form of human PRL which has potent antitumor and anti-angiogenic activities in vivo. This molecule binds to the same forms of the PRL receptor (PRLR) as unmodified PRL, yet this binding results in different intracellular signaling and biological end points. Since it is now clear that PRLRs are predimerized and therefore that ligand binding must initiate signaling by inducing a conformational change in the receptor dimer, we hypothesized that S179D PRL had an altered conformation compared to unmodified PRL. The conformation of the ligand-receptor ternary complex would therefore also have an altered conformation, and thus, different signaling molecules would be activated. Here we present evidence in support of this hypothesis by demonstrating, in contrast to unmodified PRL, that S179D PRL has reduced nickel and zinc binding capacity and a higher affinity for heparin and DEAE. Conformational changes have occurred since these features are counterintuitive on the basis of the simple substitution of a serine with a negatively charged aspartate residue. To demonstrate that these particular properties of S179D PRL were not due to misfolding of the molecule during production, S179D PRL was expressed in two different mammalian cell lines. Also investigated was the potential for production of S179D PRL as a soluble cytoplasmic, or secreted periplasmic, protein in Escherichia coli.
Ueda
Eric K M
EK
Biotechnology Department, IPEN-CNEN, Cidade Universitaria, São Paulo 05508-900, Brazil.
Soares
Carlos R J
CR
Bartolini
Paolo
P
DeGuzman
Ariel
A
Lorenson
Mary Y
MY
Walker
Ameae M
AM
D001483:Base Sequence; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005057:Eukaryotic Cells; D006801:Humans; D018716:Molecular Mimicry; D016296:Mutagenesis; D010766:Phosphorylation; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
D017931:DNA Primers; D011388:Prolactin