S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist.

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.

IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+ T cells.

Differences in the immunological reactivity of umbilical cord (UC) and adult peripheral blood (APB) T cells are poorly understood. Here, we show that IL-7, a cytokine involved in lymphoid homeostasis, has distinct regulatory effects on APB and UC lymphocytes. Neither naive nor memory APB CD4(+) cells proliferated in response to IL-7, whereas naive UC CD4(+) lymphocytes underwent multiple divisions. Nevertheless, both naive and memory IL-7-treated APB T cells progressed into the G(1b) phase of the cell cycle, albeit at higher levels in the latter subset. The IL-7-treated memory CD4(+) lymphocyte population was significantly more susceptible to infection with an HIV-1-derived vector than dividing CD4(+) UC lymphocytes. However, activation through the T cell receptor rendered UC lymphocytes fully susceptible to HIV-1-based vector infection. These data unveil differences between UC and APB CD4(+) T cells with regard to IL-7-mediated cell cycle progression and HIV-1-based vector infectivity. This evidence indicates that IL-7 differentially regulates lymphoid homeostasis in adults and neonates.

Biological properties of human prolactin analogs depend not only on global hormone affinity, but also on the relative affinities of both receptor binding sites.

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.

Growth hormone (GH) and prolactin receptors in human peripheral blood mononuclear cells: relation with age and GH-binding protein.

GH receptors (GHRs) and PRL receptors (PRLRs) were studied in human peripheral blood mononuclear cells (PBMC) using flow cytometry, biotinylated anti-GH receptor monoclonal antibody 10B8, and biotinylated human PRL. Variations of GHR and PRLR expression and the relationship of plasma GHBP and GH receptor in PBMC subsets were examined as a function of age and sex. By double immunofluorescence staining, we show that about 30% of total cells express GH receptors, with a low expression in T cells, whereas almost all B cells and monocytes are GH receptor positive. Four age groups were defined among the 64 normal volunteers, aged 12 to 85 yr, who were included in the study. The percentage of PBMC expressing GH receptors is significantly lower in group 2 (20-40 yr) than in group 1 (12-20 yr) and group 4 (>60 yr). In T cells, monocytes and B cells, no significant changes are detected in either the percentage of GH receptor positive cells or in the GH receptor level per cell. The level of PRLRs expressed in PBMC is significantly higher in age group 2 than in age group 4. A negative correlation is observed between plasma GHBP and the percentage of PBMC expressing GH receptors. These results suggest that regulation of GH receptors in lymphocytes and in other target cells could be different.

Antagonistic properties of human prolactin analogs that show paradoxical agonistic activity in the Nb2 bioassay.

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors.

Characterization of lactogen receptor-binding site 1 of human prolactin.

Prolactin (PRL) binds to two molecules of PRL receptor (PRLR) through two regions referred to as binding sites 1 and 2. Although binding site 1 has been generally assigned to the pocket delimited by helix 1, helix 4, and the second half of loop 1, the residues involved in receptor binding have not yet all been precisely identified. In an earlier alanine-scanning mutational study, we identified three major binding determinants in loop 1 of human PRL (hPRL) (Goffin, V., Norman, M. & Martial, J. A.(1992) Mol. Endocrinol. 6, 1381-1392). Here we focus on the two other regions that form binding site 1, namely helices 1 and 4. Putative binding residues, selected on the basis of a three-dimensional model of hPRL constructed in this laboratory, were mutated to alanine, and recombinant hPRL mutants produced in Escherichia coli were tested for their ability to bind to the PRLR and to stimulate Nb2 cell proliferation. We thus identified nine single mutations (three in helix 1 and six in helix 4) whose effect was to reduce both binding and mitogenic activity by more than half as compared with wild-type hPRL, indicating the functional involvement of the corresponding residues. Adding these to the three binding determinants identified in loop 1, we now propose a complete picture of PRLR-binding site 1 of hPRL. As we earlier hypothesized, the binding site 1 determinants of hPRL differ from those of human growth hormone, a hPRL homolog.

Evidence for a second receptor binding site on human prolactin.

The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the resulting mutants exhibit reduced bioactivity in the Nb2 cell proliferation bioassay, the mutated residues do not appear to be functionally necessary. Next, small residues surrounding the helix 1-helix 3 interface were replaced with Arg and/or Trp, the aim being to sterically hinder the second binding site. Several of these mutants exhibit only weak agonistic properties, supporting our hypothesis that the channel between helices 1 and 3 is involved in a second receptor binding site. We then analyzed the antagonistic and self-antagonistic properties of native hPRL and of several hPRLs analogs altered at binding site 1 or 2. Even at high concentrations (approximately 10 microM), no self-inhibition was observed with native hPRL; site 2 hPRL mutants self-antagonized while site 1 mutants did not. From these data, we propose a model of hPRL-PRL receptor interaction which slightly differs from that proposed earlier for the homologous human growth hormone (hGH) (Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., and Goeddel, D. V., and Well, J. A. (1992) Science 256, 1677-1680). Like hGH, hPRL would bind sequentially to two receptor molecules, first through site 1, then through site 2, but we would expect the two sites of hPRL to display, unlike the two binding sites of hGH, about the same binding affinity, thus preventing self-antagonism at high concentrations.