S179D-Human PRL, a Pseudophosphorylated Human PRL Analog, Is an Agonist and Not an Antagonist

Abstract

For many years, our group has been involved in the development of human PRL antagonists. In two recent publications, S179D-human PRL, a human PRL analog designed to mimic a putative S179-phosphorylated human PRL, was reported to be a highly potent antagonist of human PRL-induced proliferation and signaling in rat Nb2 cells. We prepared this analog with the aim of testing it in various bioassays involving the homologous, human PRL receptor. In our hands, S179D-human PRL was able to stimulate 1) the proliferation of rat Nb2 cells and of human mammary tumor epithelial cells (T-47D), 2) transcriptional activation of the lactogenic hormone response element-luciferase reporter gene, and 3) activation of the Janus kinase/signal transducer and activator of transcription and MAPK pathways. Using the previously characterized antagonist G129R-human PRL as a control, we failed to observe any evidence for antagonism of S179D-human PRL toward any of the human PRL-induced effects analyzed, including cell proliferation, transcriptional activation, and signaling. In conclusion, our data argue that S179D-human PRL is an agonist displaying slightly reduced affinity and activity due to local alteration of receptor binding site 1, and that the antagonistic properties previously attributed to S179D-human PRL cannot be confirmed in any of the assays analyzed in this study. (Endocrinology 142: 3950 -3963, 2001)

Development of Pure Prolactin Receptor Antagonists

“…2A). We have shown previously that the hGH analog G120K-hGH is also a potent antagonist in this assay (50), whereas the analog S179D-hPRL is not an antagonist but is instead a super-agonist (42). Similar observations were confirmed in this study (not shown).…”

“…We believe that removal of the two N-terminal cysteines (Cys 4 -Cys 11 ) prevents formation of covalent multimers involving intermolecular disulfide bonding between these residues. 3 Production of S179D was less efficient, probably because of misfolding as reported previously (42).…”

“…Hormones-In this study, we used exclusively recombinant proteins as follows: WT hPRL (41), the molecular mimic of phosphorylated PRL named S179D-hPRL (42); the first generation antagonist G129R (Gly 129 replaced with Arg) (31); and the two new antagonists constructed in this study, ⌬1-9-G129R-hPRL and ⌬1-14-G129R-hPRL, characterized by the deletion of the 9 or 14 N-terminal residues, respectively. The pT7L expression vector used for expression of all hPRL analogs was described previously (41).…”

“…Proliferation studies were performed using the reference assay for lactogens, Nb2 cells (47,48), or the proliferation assay that we recently established using Ba/ F-03 cells stably transfected with the expression plasmid encoding the human PRLR (referred to as Ba/F-LP cells). The transcriptional study was based on the activation of the lactogenic hormone-response element (LHRE)-luciferase reporter gene, performed using the HL5 clone as recently described (39,42,46). Finally, we used T47D human breast cancer cell lines to assess signaling events triggered by the PRLR, using procedures described under "Signaling Studies" below.…”

S179D prolactin: Antagonistic agony!

“…However, when used alone and at high concentrations in cells that cannot activate alternate splicing of the receptor (see later), it shows some agonist activity, with about a two log increase in the amount required for equivalent activity to the unmodified hormone (Bernichtein et al, 2001;Soares et al, 2006). However, there is no circumstance in vivo when unmodified PRL is absent.…”