  Development of pure prolactin receptor antagonists; ; et al in Journal of Biological Chemistry (2003), 278(38), 35988-99 Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the ... [more ▼] Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all forms of PRL receptor antagonists obtained to date exhibit partial agonism, preventing their therapeutic use as full antagonists. In the present study, we describe the development of new human PRL antagonists devoid of agonistic properties and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including highly sensitive assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as assessed by analyzing their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland, and prostate). Finally, by using transgenic mice expressing PRL specifically in the prostate, which exhibit constitutively activated signaling cascades paralleling hyperplasia, we show that these new PRL analogs are able to completely revert PRL-activated events. These second generation human PRL antagonists are good candidates to be used as inhibitors of growth-promoting actions of PRL. [less ▲] Detailed reference viewed: 15 (1 ULg)S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist; ; et al in Endocrinology (2001), 142(9), 3950-63 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 ... [more ▼] 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. [less ▲] Detailed reference viewed: 7 (1 ULg)Molecular bases of the interaction between human prolactin and its membrane receptor: A ten year study; ; et al in Recent research developments in endocrinology (2001), 2 In this review, we make a chronologically overview of a project aimed at deciphering the molecular bases of the mechanism by which prolactin, a pituitary-secreted polypeptidic hormone, interacts with its ... [more ▼] In this review, we make a chronologically overview of a project aimed at deciphering the molecular bases of the mechanism by which prolactin, a pituitary-secreted polypeptidic hormone, interacts with its membrane receptor, a member of the cytokine receptor superfamily. This study, based essentially on structural and mutational analysis of human prolactin, has identified two regions interacting with the receptor, named binding sites 1 and 2. We have also provided evidence that prolactin induces dimerization of its receptor through its two binding sites, and based on that mechanism of activation, we have engineered and characterized prolactin antagonists able to block the effect of the hormone by competition for receptor binding. Our study has also highlighted the importance of using well characterized bioassays to monitor such a structure-function study since the properties of a given hormone analog were shown to strongly differ depending on the bioassay used. [less ▲] Detailed reference viewed: 15 (4 ULg) Biological properties of human prolactin analogs depend not only on global hormone affinity, but also on the relative affinities of both receptor binding sites; ; et al in Journal of Biological Chemistry (1999), 274(37), 26033-43 Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174(hGH) and His-18(hGH). In contrast, binding of ... [more ▼] Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174(hGH) and His-18(hGH). In contrast, binding of hPRL to the hPRLR is zinc-independent. We engineered in binding site 1 of hPRL a hGH-like zinc coordination site, by mutating Asp-183(hPRL) (homologous to Glu-174(hGH)) into Glu (D183E mutation). This mutation was also introduced into G129R hPRL, a binding site 2 mutant (Goffin, V., Kinet, S., Ferrag, F., Binart, N., Martial, J. A. , and Kelly, P. A. (1996) J. Biol. Chem. 271, 16573-16579). These analogs were characterized using a stable clone expressing both the hPRLR and a PRLR-responsive reporter gene. The D183E mutation per se decreases the binding affinity and transcriptional activity of hPRL. However, this loss is partially rescued by the addition of zinc and the effect is much more marked on bioactivity than on binding affinity. These data indicate that the D183E mutation confers zinc sensitivity to hPRL biological properties. Due to an impaired site 2, the agonistic activity of G129R analog is almost nil. Although the double mutant D183E/G129R displays lower affinity ( approximately 1 log) compared with G129R hPRL, it unexpectedly recovers partial agonistic activity in the absence of zinc. Moreover, whereas zinc increases the affinity of D183E/G129R, it paradoxically abolishes its agonistic activity. Our results demonstrate that the biological properties of hPRL analogs do not necessarily parallel their overall affinity. Rather, the relative affinities of the individual binding sites 1 and 2 may play an even more important role. [less ▲] Detailed reference viewed: 6 (0 ULg) |
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