Isabelle Dusanter-Fourt

The interaction of prolactin with its receptors in target tissues and its mechanism of action.

Publisher Summary This chapter describes the interaction of prolactin with its receptors in target tissues and with its mechanism of action. Prolactin is a hormone secreted by the anterior pituitary gland, whose primary action involves the development of the mammary gland and production of milk, which is essential for the survival of newborn mammals. The hormonal regulation of prolactin receptors is complex. Prolactin receptors have been localized in a number of different tissues, although they have been most extensively studied in the mammary gland and liver. Receptor numbers differ dependent upon the physiological state of the animal. A large percentage of prolactin receptors are located within the cell in the Golgi and lysosomal compartments. Prolactin can regulate its receptor number both in a positive and negative fashion. A slow upregulation is observed most generally in vivo following extended periods of injection, corresponding to a large extent, to the accumulation of Golgi membranes that occurs during differentiation of the mammary gland. In rat liver cells in suspension culture, this upregulation represents a reduced level of degradation as well as an increased synthesis of receptors. The downregulation of prolactin receptors is a more rapid and reversible process. It can be observed both in vivo and in vitro in rabbit mammary glands and rat liver.

In vivo lactogenic effects of anti prolactin receptor antibodies in pseudopregnant rabbits

Antibodies generated against partially purified prolactin receptors from rabbit mammary gland membranes were tested for their effects on prolactin binding to receptors and for their in vivo biological potencies. These antibodies are able to inhibit prolactin binding to crude rabbit mammary gland membranes. When administered intravenously or intramuscularly to pseudopregnant rabbits, they induce respectively an accumulation of beta-casein or an enhancement of beta-casein synthesis and mRNA concentration in the mammary gland. Moreover the stimulatory effect of these anti-prolactin receptor antibodies on casein synthesis is totally abolished by a simultaneous treatment with progesterone, which is a potent in vivo inhibitor of prolactin action. These results better establish the prolactin-like activities of these antibodies previously observed in vitro and give strong support to the hypothesis that prolactin molecule is not required beyond the initial binding to its receptor to induce hormonal effects.

Purification of prolactin receptors from sow mammary gland and polyclonal antibody production

After solubilization with Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), prolactin receptors from mammary crude membranes of primiparous lactating sows (pretreated with bromocriptine) have been purified by affinity chromatography using ovine prolactin or a monoclonal antibody against rabbit prolactin receptor. Comparative analysis of these two methods of purification demonstrated that use of an immunoaffinity step allowed a great improvement of receptor yield (40%) compared to the hormone affinity method (10%). In addition, partially purified fractions obtained by immunoaffinity appeared more homogeneous and had much higher specific activity. Affinity labelling of prolactin receptors from crude membranes or solubilized extracts with iodinated ovine prolactin, followed by electrophoretic analysis (SDS-PAGE) and autoradiography, revealed one binding unit of approximately 45 kDa. When partially purified receptor preparations were labelled with 125I, submitted to an additional affinity chromatography and analyzed by SDS-PAGE, prolactin receptors appeared as a single form having a molecular weight of 42-45 kDa, which is not associated with itself or other subunits by disulfide linkages. Partially purified fractions were used to produce anti-prolactin receptor serum from goats. These polyclonal antibodies were able to completely inhibit the binding of lactogenic hormones in sow and rabbit mammary membranes. They were also able to recognize hormone-receptor complexes, but more specifically in sow mammary gland. These antisera could inhibit prolactin binding to its receptors in several organs of various species, suggesting that prolactin receptors shared numerous antigenic similarities between species and particularly between sow and rabbit. These similarities appeared to be located essentially on the part of the molecule more specifically involved in the recognition of the hormone.

Effects of transglutaminase or phospholipase A2 inhibitors on down-regulation of prolactin receptors and stimulation of casein and DNA synthesis in mammary gland explants

A number of compounds have been found to inhibit the internalization of alpha 2-macroglobulin and/or epidermal growth factor into fibroblasts. Using the same inhibitor we tried to block the down-regulation of prolactin receptors, supposed to mirror internalization of PRL receptors, in order to investigate the possible role of internalization in the mechanism of prolactin action. In rabbit mammary cells it appeared that bacitracin and ethylamine completely block prolactin receptor down-regulation, but dansylcadaverine, the most potent inhibitor of transglutaminase, was without effect. The blockage of phospholipase A2 by chlorpromazine or bromophenacyl bromide (BPB) was without effect on the down-regulation of prolactin receptor. Subcellular distribution of [125I]oPRL has been studied after incubation with or without these various inhibitors after fractionation of mammary gland homogenate on sucrose gradient. Any of these compounds was able to increase the labelling of prolactin receptors located in plasma membrane fractions, suggesting that the rate of internalization of prolactin was not modified. In addition, none of these compounds inhibited the stimulation of beta-casein and DNA synthesis by prolactin. These results suggest that both transglutaminase and phospholipase A2 are not involved in the mechanism of prolactin-induced down-regulation of prolactin receptors, although bacitracin and ethylamine are able to block this phenomenon probably by different mechanism. In all cases, inductions of mitogenesis and beta-casein synthesis by prolactin in the rabbit mammary cells were not modified by the various compounds utilized. We conclude that neither transglutaminase nor phospholipase A2 are involved in the internalization of prolactin-receptor complexes, although bacitracin, ethylamine and quinacrine are able to block the down regulation of prolactin receptors by other means.

Prolactin receptor: identification of the binding unit by affinity labelling and characterization of poly- and monoclonal antibodies

The prolactin receptor localized in rabbit mammary gland membranes has been identified by affinity labelling using covalent cross-linking agents such as a unique protein chain of approximately 32,000 daltons. After partial purification (5,000-fold) of these receptors from mammary gland homogenate, polyclonal antibodies, which specifically and completely inhibit prolactin binding in all organs and in all species studied, were raised. These antibodies possessed prolactin-like biological activity (casein synthesis) on rabbit mammary gland explants. Monoclonal antibodies specifically directed against the binding domain of the receptor were also obtained. These antibodies were more species-specific than the polyclonal antibodies. The most potent (M110) possessed higher affinity than prolactin for the receptor and could be a very effective tool to elucidate the structure of the receptor and its immunological detection.

PARTIAL PROTEOLYTIC DIGESTION OF THE MAMMARY PROLACTIN RECEPTOR : IDENTIFICATION OF SMALLER PROLACTIN BINDING FRAGMENTS

Partial proteolytic digestion of the mammary prolactin (PRL) receptor was used to generate receptor fragments and analyze their immunoreactivity and PRL binding properties. Tryptic digestion of the PRL receptor produced two immunoreactive fragments (Mr approximately 30,000 and approximately 15,000) that reacted with a monoclonal anti-PRL receptor antibody and still specifically bound PRL, while the complete immunoreactive PRL binding unit (Mr approximately 42,000) disappeared. Neither chymotrypsin nor V8 protease were able to generate any immunoreactive receptor fragments. These receptor fragments may represent smaller PRL binding receptor form(s) of biological significance.

Characterization of antisera to prolactin receptors

Publisher Summary This chapter explains that techniques were designed to improve purified prolactin (PRL) receptors and immunize several species of animals with the partially purified receptor to increase the yield of antisera and better characterize the specificity of these antisera in different target organs. The action of PRL, as is true for other peptide hormones, is mediated by a specific receptor located in membrane components of the cell. The PRL receptor has been well characterized and shown to respond to both a down- and an up-regulation by the hormone. Antibodies to the PRL receptor have been shown to inhibit the capacity of prolactin to support the synthesis of casein and the uptake of α-isoaminobutyric acid by rabbit mammary explants in culture and attenuate the action of prolactin on mammary gland and ovary when injected into rats. Anti-prolactin receptor serum is able to both inhibit and mimic the action of prolactin in rabbit mammary explants and in rat liver.