Development of a constitutively active mutant form of the prolactin receptor, a member of the cytokine receptor family.

The extracellular domain of the PRL receptor (PRL-R) is composed of two subdomains of approximately 100 amino acids, S1 and S2. To explore the functional significance of these subdomains in PRL binding and signal transduction, deletion mutants of S1 or/and S2 subdomains were constructed. We report here the inability of each of these mutant receptor forms to bind PRL after expression in COS-7 cells. We also studied the abilities of these different mutant receptors to respond to hormonal stimulation after transfection of each mutant complementary DNA into CHO-K1 cells along with a chimeric gene containing the promoter of a milk protein gene (beta-lactoglobulin) fused to chloramphenicol acetyltransferase coding sequence. Somewhat unexpectedly, a constitutively (PRL-independent) mutant form of the PRL-R was obtained after deletion of the S2 subdomain. Moreover, we analyzed, in CHO-K1 cells, the biological activity of chimeric receptors constructs in which each subdomain sequence was replaced by an unrelated, but coding, sequence of foreign protein, and we confirmed a specific requirement for the S1 sequence in the constitutive activity. In contrast, the S2 subdomain produced an inhibitory effect on S1 constitutive activity. Cotransfection experiments with the wild-type receptor and the constitutive mutant receptor provided evidence that the wild-type receptor was able to inhibit the constitutive activity of the deleted mutant. Furthermore, in the mouse mammary epithelial cell line HC11, the constitutive PRL-R form was able to induce transcription of the beta-casein gene in the absence of PRL. These results suggest a complex signal transduction process that implicates each extracellular PRL-R subdomain. Possible mechanisms for the constitutive effect are discussed.

Gonadotropins in lactating sows exposed to long or short days during pregnancy and lactation: serum concentrations and ovarian receptors.

The objective of this study was to determine the influence of day length on gonadotropin profiles and the expression of their ovarian receptors in lactating sows. Primiparous Large White gilts were exposed to either a gradual increase (from 12 to 16 h/day, LONG treatment, n = 13) or decrease (from 12 to 8 h/day, SHORT treatment, n = 12) in photoperiod during gestation. Weaning occurred at Day 21 of lactation. All 4 sows that were submitted to the SHORT light duration and checked for postpartum estrus demonstrated an estrus by 10 days postwearing in contrast to 2 of 5 sows submitted to the LONG light duration (p < 0.05). In the remaining 16 sows, day length had no significant effect on the number of LH pulses or on mean or basal concentrations of gonadotropins (FSH and LH) or estradiol-17 beta measured at Day 20 of lactation. Ovarian receptors for gonadotropins and prolactin (PRL) and their mRNA were measured through use of receptor-binding and slot-blot analyses, respectively, at Day 21 of lactation in these 16 sows. ALONG photoperiod duration had no influence on receptor number, binding or affinity, but it significantly increased LH receptor mRNA levels (p < 0.05). However, FSH receptor mRNA levels were similar in the two groups of sows. Plasma LH concentration was positively related to LH and FSH receptor content but not to their cognate mRNA levels. Plasma concentration of FSH was positively related to the level of its own receptor mRNA as well as to that of the PRL receptor mRNA. Although the LONG day length may have delayed the return to estrus, there was no effect on gonadotropin secretion. Our results show an effect of photoperiod only on the level of LH receptor mRNA. We suggest that not all transcripts of the LH receptor are translated and that nontranslatable mRNA accumulate in ovaries of sows exhibiting delayed estrus.

Prolactin induces growth inhibition and promotes differentiation of CHO cells stably transfected with prolactin receptor complementary DNA.

We have characterized a stable and functional transfectant of the rabbit prolactin receptor in Chinese hamster ovary cells, and investigated the action of prolactin (PRL) on the growth and differentiation of this transfectant (clone E32). PRL induced a significant inhibition of E32 cell proliferation. Growth inhibition correlated with gene induction of the molecular marker of ovarian differentiation cholesterol side chain cleavage P450 (P450scc). Both effects were inversely proportional to cell confluence. The limits and potential development of such transfected cellular systems are discussed.

Effects of anoestrus and bromocryptine treatment on the expression of prolactin and LH receptors in the rabbit ovary during lactation.

The aim of the present study was to correlate the number of prolactin and LH receptors in the ovary with the changes in sexual behaviour that occur within a few days following parturition in rabbits. Multiparous New Zealand white rabbits at days 0, 3 and 10 of lactation were tested for their receptivity upon presentation to a male. Rabbits were classed as either receptive or nonreceptive at each stage of lactation; half of the animals in each class were treated with bromocryptine to examine the effects of prolactin deprivation. Ovarian receptors for LH and prolactin, as well as the concentration of their corresponding mRNA, were measured at each stage of lactation in every group. Results indicate that receptive behaviour is correlated with significantly more follicles on the rabbit ovary (diameter > 1 mm; P < 0.05) and an increase in the concentration of LH receptor mRNA (P < 0.001) and prolactin receptors (P < 0.05). In addition, on day 4 of lactation, there were significantly fewer follicles in nonreceptive rabbits (P < 0.05). LH receptor content remained constant on days 1 and 4 of lactation but increased on day 11 (P < 0.05). Bromocryptine treatment had no effect on the number of follicles or on the amount of LH receptor mRNA in does, but it significantly increased LH receptors (P < 0.01), and the concentration of prolactin receptor mRNA (P < 0.001), particularly on day 11 of lactation (P < 0.05), and prolactin receptor content (P < 0.001). Receptive rabbit ovaries therefore display more follicles that can respond to an LH surge via newly transcribed LH receptors than do nonreceptive.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and regulation of the angiotensin II type-1 receptor (binding and mRNA) in human adrenal fasciculata-reticularis cells.

The classical concept of human adrenal physiology indicates that only glomerulosa cells are the target of A-II. Herein, we demonstrated that cultured human adrenal fasciculata-reticularis cells were also responsive to this hormone. Indeed, these cells contained high affinity (Kd = 0.9-1.1 nM) and low capacity (8,000-13,000 sites/cell) A-II receptors, and more than 95% of them were of the type-1. These AT1 receptors are functional since A-II was able to increase cortisol production after 48 h of treatment. These effects were inhibited by losartan, an AT1 antagonist, but not by CGP42112A, an AT2 antagonist. The expression of the type-1 A-II receptor mRNA was detected in the whole adrenal in both adult and fetus, and in cultured human adrenal fasciculata-reticularis cells. In these cells A-II negatively regulated AT1 receptor mRNA, and this effect was also mediated through the AT1 receptor subtype.

Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development.

The yellow (y) gene of Drosophila is required for the formation of black melanin and its deposition in the cuticle. We have studied by immunohistochemical methods the temporal and spatial distribution of the protein product of the y gene during embryonic and pupal development and have correlated its expression with events of cuticle synthesis by the epidermal cells and with cuticle sclerotization. Except for expression in early embryos, the y protein is only found in the epidermal cells and may be secreted into the cuticle as it is being deposited. The amount of y protein in various regions of the embryo and pupa correlates directly with the intensity of melanization over any section of the epidermis. Expression of the y gene begins in the epidermal cells at 48 hr after pupariation and is well correlated with the beginning deposition of the adult cuticle. At this stage the adult cuticle is unsclerotized and unpigmented and dopa decarboxylase levels, a key enzyme in catecholamine metabolism which provides the crosslinking agents as well as the precursors for melanin, is low. As a separate event 26 hr after the onset of y gene expression, the first melanin deposition occurs in the head bristles and pigmentation continues in an anterior to posterior progression until eclosion. This melanization wave is correlated with elevated dopa decarboxylase activity. Crosslinking of the adult cuticle also occurs in a similar anterior to posterior progression at about the same time. We have shown by imaginal disc transplantation that timing of cuticle sclerotization depends on the position of the tissue along the anterior-posterior axis and that it is not an inherent feature of the discs themselves. We suggest that actual melanization and sclerotization of the cuticle by crosslinking are initiated at this time in pupal development by the availability of the catecholamine substrates which diffuse into the cuticle. Intensity of melanization and position of melanin pigment is determined by the presence or absence of the y protein in the cuticle, thus converting the y protein prepattern into the melanization pattern.