Binding Of 125I-labeled Ovine Prolactin Research Articles

Identification of ligand binding determinants of the prolactin receptor.

The prolactin (PRL) receptor, a lactogen- and primate somatogen-binding protein, is a member of an expanding superfamily (cytokine/growth hormone (GH)/PRL) of single membrane-spanning receptors. Two features commonly shared among this group of proteins are the presence of two pairs of cysteines, generally found in the N-terminal region of the extracellular domain, and a WSxWS (WS) motif, frequently located proximal to the transmembrane domain. We have recently shown the 4 cysteines to be critical to the maintenance of the structural and functional integrity of the PRL-receptor. In the present study, we prepared a set of eight chimeric rat PRL/human GH receptors and several alanine mutants, to assess the importance of the Cys-rich domain (residues 12-68) in confering specificity to PRL binding. The role of the WS motif in high affinity binding was also investigated. Binding of 125I-labeled ovine PRL or human GH to membrane preparations from COS-7 cells transiently expressing the mutant receptors have defined a region within the first disulfide loop (residues Arg13, Asp16, Glu18) and the set of lactogen-specific sequences between the two pairs of cysteines as key determinants of PRL-binding specificity, which converge to form a patch on a two-dimensional model of the PRL receptor. We also demonstrate that, although PRL- and GH-specific determinants overlap in certain areas, they are not identical. Finally, substitution of the WS motif with alanine residues precludes high affinity binding to ovine PRL and human GH and suggests that this structural element may provide a target site for the interaction of an accessory protein necessary for the formation of a high-affinity receptor complex.

Partial Purification of Prolactin-Like Substance from Snake(Ptyas Mucosa) Pituitaries.

Pituitary extract of the common rat snake (Ptyas mucosa) was found to be capable of displacing the binding of 125I-labelled ovine prolactin to female rat liver membranes, suggesting the presence of prolactin-like substance in snake pituitary. The snake prolactin-like substance was unadsorbed on Concanavalin A-Sepharose, but adsorbed on DEAE-cellulose. The partially purified snake prolactin-like substance was also capable of displacing the binding of 125I-labelled ovine prolactin to snake kidney and large intestine membranes. Chromatographic fractions derived from snake pituitary and which possessed potent growth hormone receptor binding activity were devoid of prolactin receptor binding activity, suggesting the existence of distinct prolactin-like and growth hormone-like substances in snake pituitary.

Further study on the changes in the concentration of prolactin-binding sites in different organs of Xenopus laevis male and female, kept under dry conditions and then returned to water (their natural habitat)

The binding of 125I-labeled ovine prolactin (125I-oPRL) to membranes from the kidney and liver of Xenopus laevis male and female specimens (Experiment I) and from the kidney, epidermis, and liver of female specimens (Experiment II) (1) kept in an aquatic environment, (2) exposed for 2 weeks to dehydrating conditions, and (3) then placed back into their aquaria after exposure to dehydrating conditions (Experiment II) was studied. No significant sex differences in PRL binding to kidney, epidermis, and liver were found. A highly significant drop in PRL specific binding to the membranes from the kidney and epidermis is brought about in the specimens from both sexes exposed to dehydrating conditions. The results obtained by MgCl2 treatment in vitro of the membranes under study for an estimation of total PRL receptor concentrations seem to point to an actual decrease in PRL specific binding sites. The values of PRL specific binding to the membranes from the liver are not affected by dehydration of the animals (Experiment I and II) or their subsequent rehydration (Experiment II). In rehydrated females (Experiment II), PRL binding values were closely related to those recorded in females permanently maintained in water (controls).

Regional distribution and species variation of prolactin binding sites in the brain

The binding of 125I-labeled ovine prolactin ( 125I-oPRL) to membranes from different brain regions of pigeon, rabbit, rat, pig, calf, horse, and ewe was studied. The hypothalamus from rabbit, pig, horse, and pigeon showed a low but specific binding for 125I-oPRL clearly different from the other brain regions examined (cortex and cerebellum), whereas in the brain from rat, calf, and ewe the binding was very small and quite uniform in the various regions. Also the membranes from choroid plexus of rabbit, pig, calf, and horse showed an evident specific binding for prolactin. The binding of 125I-oPRL to hypothalamus and choroid plexus membranes from rabbit and horse was inhibited in a dose-dependent manner by unlabeled oPRL and hGH but not by many other polypeptide hormones. Scatchard analysis of the binding revealed the presence of binding sites with low capacity and high affinity for ovine ligard.

Prolactin receptors in the male Rana esculenta

The binding of 125I-labeled ovine prolactin (oPRL) to membrane preparations of several tissues from the male green frog, Rana esculenta, collected during the year is reported. PRL binding to kidney fractions was generally high (range 5–45%). A maximum was observed in the month of October, whereas the lowest value was found during the summer season. The binding to skin fractions was equally high (range 5–25%) and the annual profile parallels that of renal fractions. In the liver, a lower specific binding (range 3–4%) occurred consistently during the year, whereas no detectable binding was found in the muscle. The 125I-oPRL binding was inhibited by oPRL and oGH but not by oFSH or oLH. Scatchard analysis gave dissociation constants of 0.4–1 × 10 −10 M and binding capacity of about 20 fmol/mg of membrane proteins was observed in both the skin and kidney fractions. No receptor sites were detectable in 30-day hypophysectomized animals. The administration of oPRL or a crude homogenate of the frog hypophysis induced the appearance of specific PRL binding. Testosterone is able to restore prolactin binding in hypophysectomized animals, as PRL treatment does.

Prolactin binding sites in Xenopus laevis tissues: Comparison between normal and dehydrated animals

The binding of 125I-labeled ovine prolactin ( 125I-oPRL) to membranes from the kidney, epidermis, liver, and testis of Xenopus laevis adult speciments either kept in an aquatic environment or exposed for 2 weeks to dehydrating conditions was studied. Prolactin binding specificity was assayed through competition with several unlabeled hormones (oPRL, hGH, rGH, rLH, and porcine insulin). In the animal exposed to dehydrating conditions a statistically highly significant reduction in prolactin binding to the membranes from the kidney and epidermis was recorded. No significant variations were revealed by the membranes from the liver and testis. The reduction detected in the binding of 125I-oPRL is not related to the dissociation constant, but to the number of PRL binding sites. Since PRL ranks among the few peptide hormones whose rise in the bloodstream promotes an increase in the number of their own receptors, the reduction of its binding sites in Xenopus specimens exposed to dehydration might lend some support to our earlier hypothesis that transfer to a dehydrating environment may bring about, in this totally aquatic species, some decrease in the blood PRL levels.

Prolactin receptors in tilapia ( Sarotherodon mossambicus) tissues: Binding studies using 125I-labeled ovine prolactin

The binding of 125I-labeled ovine prolactin (oPRL) to membrane preparations of tissue from freshwater-adapted tilapia ( Sarotherodon mossambicus) was examined. Liver, ovary, and testis showed a relatively high specific binding (5–10%). A lower specific binding occurred consistently in intestine and gill tissue, and inconsistently in urinary bladder and kidney preparations. Desaturation experiments with MgCl 2 indicated that a majority of the PRL receptors were already occupied by endogenous PRL. Scatchard analysis of liver binding gave a dissociation constant of 0.6 × 10 −9 M and a capacity of 207 fmol/mg protein.

Properties of hepatic binding sites for prolactin in ring doves ( Streptopelia risoria)

Specific binding of 125I-labeled ovine prolactin ( 125I-oPRL) was detected in crude membrane fractions prepared from ring dove ( Streptopelia risoria) liver homogenates. In characterization studies, specific binding was found to depend upon pH, incubation time, incubation temperature, and membrane protein concentration. Competitive inhibition of specifically bound 125I-oPRL was observed with human growth hormone, human and rat prolactin, and dove pituitary extract but not with turkey prolactin, human placental lactogen, and several nonlactogenic hormone preparations. Dove liver membranes showed high affinity ( K d = 3 × 10 −10 M) for binding to oPRL but had relatively low binding capacity ( B max < 20 fmol/mg protein). PRL binding activity in pooled liver fractions from breeding doves during early stages of incubation prior to crop sac growth did not differ markedly from that observed in doves sampled at the end of incubation when crop sac weight and serum PRL were elevated. However, binding activity was higher in pooled male liver fractions than in pooled female liver fractions at both reproductive stages. A two- to threefold increase in binding capacity was observed in pooled liver fractions from late-incubating doves following MgCl 2-induced binding site desaturation. The MgCl 2 treatment did not eliminate the differences in specific binding observed between male and female liver fraction pools, thus suggesting the possibility of sex-specific mechanisms of hepatic PRL binding site regulation in this species. It is concluded that the dove liver possesses specific binding sites for PRL with properties similar, but not identical, to those found in other vertebrate target tissues.

Characterization of antisera to a partially purified prolactin receptor: effect on prolactin binding in different target tissues

Prolactin (PRL) receptors have been purified by affinity chromatography using a lactogenic hormone (human growth hormone, hGH) coupled to Affigel-10. The mean binding capacity of 3 separate purifications was 1.18 ± 0.26 moles prolactin per mg protein, representing a 836-fold purification over crude microsomes and 4000−5000-fold over mammary gland homogenates and 16% purity. The receptor was characterized by HPLC using a TSK-SW-4000 and 3000 column connected in series and eluted in 0.1 M borate buffer containing 0.2 M NaCl and 0.1% Triton. A single peak of [ 125I]hGH binding activity with a retention time of 45.2 min (22.6 ml), representing an apparent molecular weight of 133000, was observed. A single peak of activity was also observed following polyacrylamide gel electrophoresis of the purified receptor in 0.1% Triton coincident with the Coomassie-stained protein band. Antibodies to the partially purified receptor preparations were prepared in sheep, goats and guinea pigs. Antisera to the prolactin receptor prepared in all three species were capable of inhibiting the binding of 125I-labeled ovine prolactin to receptors from rabbit mammary glands. Significant inhibition of binding was observed at antisera dilution of 1:10 000 with the sheep antiserum being the most potent (half-maximal inhibition (IM 50) = 1:5700). All three antisera were able to inhibit PRL binding completely, but failed to affect labeled insulin or hCG binding and had very little effect on bGH binding. The specificity of the sheep antiserum was tested in rabbit mammary glands, ovary, adrenal, pig mammary glands and 8 rat tissues which contain PRL receptors. The antiserum was able to inhibit the binding of labeled PRL in all tissues, with the inhibition curves for the rat tissues being non-parallel when compared to rabbit mammary gland, suggesting a homology but not a complete identity between PR1 receptors in various tissues and animal species. These studies demonstrate that prolactin receptors can be purified from rabbit mammary tissue and that antisera can be produced in several species. In addition, the binding studies suggest that in the various tissues the receptor molecule is more or less exposed to interaction with the antisera, or that the receptor protein differs somewhat between species.

Binding of oestradiol, progesterone and prolactin in rat lung.

Binding of [3H]oestradiol and of [3H]progesterone in the cytosol from lungs of adult male rats was suppressible, dependent on incubation time and on protein concentration and was protein in nature. Suppressible binding of oestradiol consisted of a high affinity site (dissociation constant (Kd), 1 x 10(-9) +/- 0.2 x 10(-9) mol/l; maximum number of binding sites (Bmax), 0.7 +/- 0.2 pmol/mg protein) and a lower affinity site (Kd, 2.4 x 10(-8) +/- 0.6 x 10(-8) mol/l; Bmax, 6.3 +/- 0.4 pmol/mg protein) and showed evidence of positive co-operation. Suppressible binding of progesterone consisted of a single site with a Kd of 6 x 10(-9) +/- 1 x 10(-9) mol/l and Bmax of 44.5 +/- 8 fmol/mg protein. Binding of 125I-labelled ovine prolactin was found in homogenates of fetal lung (20 days of gestation) but not of adult lung (80 days of age). Treatment of adult rats with ovine prolactin was associated with an increase in the number of binding sites of high affinity for 125I-labelled ovine prolactin but these sites showed an altered specificity. This 'up-regulation' of the prolactin binding may provide a mechanism by which prolactin stimulates surfactant production in lung. These results, together with the known effects of these hormones on certain lung functions, provide further evidence that lung is a target organ for oestradiol, progesterone and prolactin.

Prolactin binding sites on human chorion-decidua tissue

An effective procedure has been developed and utilized to demonstrate the presence of prolactin receptors on the plasma membranes of human chorion-decidua cells. Particulate fractions from human chorion-decidua sedimenting between 1,500 and 45,000 × g display optimal binding of 125I-labeled ovine prolactin when incubated at a membrane protein concentration of 200 μg per assay tube for 2 hours at 22° C. Specific binding was increased by pretreatment of the membrane particles with 5M magnesium chloride to remove endogenous prolactin. These receptors show binding parameters (affinity, 0.92 × 109 L/mole; capacity, approximately 80 fmoles/mg) similar to those of lactogenic receptors in the rabbit mammary gland and, the rabbit and rat liver. The presence of prolactin receptors in human chorion-decidua suggests they may play a role in mediating local action(s) of prolactin such as involvement in the decidualization reaction or in maintaining fetal osmoregulation.

Kinetic properties of rat hepatic prolactin receptors

Binding of 125I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K 1, of 2.1 × 10 7, 1.4 × 10 7, 1.2 × 10 7 and 4 × 10 6 M −1. min −1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K −1=1.26 × 10 −2. min −1 ( T 1 2 =55 minutes ) and a slower phase with K −1=1.103 × 10 −3. min −1. The apparent K d (from K −1K 1) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.

Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.

Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and casein mRNA accumulation as a function of the antiserum concentration. However, in the absence of prolactin, both antisera (guinea pig and sheep) at moderate concentrations were capable of mimicking prolactin action on casein gene expression and on DNA synthesis. At higher concentrations, the anti-prolactin receptor antibodies inhibited their own actions. Several characteristics of the prolactin effect were also observed with the anti-prolactin receptor antibody: the stimulatory effect of the antibody was amplified by glucocorticoids; colchicine, which was capable of blocking prolactin action, also prevented the induction by the antibody. Lysosomotropic agents, which do not interfere with prolactin action, did not alter the response observed with the antibody. These results indicate that an anti-prolactin receptor antibody can mimic two major actions of prolactin obtained in mammary explant culture and suggests that the prolactin molecule is not required beyond the initial binding to its receptor.

Effect of prolactin and prostaglandins on the stimulation of prolactin binding sites in the male rat liver

Induction of prolactin hepatic receptors in the male rat by exogenous prolactin and the possible involvement of prostaglandins in this process were studied. When ovine prolactin 500 μg/kg was administered sc in saline containing 10% PVP (polyvinylpyrrolidone), twice daily for 6.5 days and the rats killed 48 h after the last injection, the specific binding of 125I-labelled ovine prolactin to prolactin hepatic receptors was raised 26-fold, while administration of prolactin in saline only caused a 7-fold increase. A well correlated log dose-response relationship was demonstrated between 12.5 and 500 μg of prolactin in saline-PVP, with the lowest dose causing an 18-fold increase in binding. A shorter 4.5 day treatment of prolactin in saline—PVP caused only a small 3-fold increase in prolactin binding. Scatchard analysis showed that these increases resulted from increases in receptor concentration. The effect of prolactin on the induction of the hepatic receptors could not be mimicked by PGE 1, PGE 2 or PGF 2α, nor could PGF 2α synergize with a short treatment with prolactin. Further, indomethacin caused no significant effect on this action of prolactin. It seems that prolactin does not induce its own receptors in the rat liver by stimulation of prostaglandins in this tissue.

Prolactin receptors in the rat kidney.

Specific binding of 125I-labelled ovine prolactin iodinated by a lactoperoxidase method was demonstrated in crude membrane preparations of kidneys and adrenals of male Sprague-Dawley rats and livers from female rats. Membrane preparations derived from the 100,000 g fractions of tissue homogenates contained most of the specific prolactin binding. Kinetic and affinity characteristics of prolactin binding to kidney membranes were examined in detail. Maximal specific binding occurred after incubation for 30 h at room temperature. Scatchard analysis indicated that prolactin binding to kidney membranes was of high affinity (dissociation constant = 1.4 x 10(-10) mol/l) and similar to that for liver membranes, although kidney membranes from male rats bound approximately sixfold less prolactin/mg membrane protein than did liver membranes from female rats. Specific prolactin binding was demonstrated in both renal medulla and cortex. Autoradiography showed maximal prolactin binding activity in the epithelial cells of the proximal tubule and faint activity in the tubular cells throughout the nephron. Specificity of uptake by proximal tubular cells was indicated by the gross reduction in prolactin activity when excess ovine prolactin was administered simultaneously. The demonstration of specific binding sites for prolactin localized primarily in the proximal tubules was consistent with renal action of prolactin, predominantly on sodium metabolism.

Prolactin-binding sites in tilapia ( Sarotherodon mossambicus) kidney

Highly purified 125I-labeled tilapia ( Sarotherodon mossambicus) prolactin (PRL) binds to microsomal membrane fractions prepared from tilapia kidney but not liver or gill. Specificity studies revealed that up to 35% of the labeled tilapia PRL bound to the kidney membrane preparation could be displaced by 100 ng of unlabeled hormone. Ovine PRL displaced 125I-labeled tilapia PRL from the kidney PRL-binding site. However, 125I-labeled ovine PRL demonstrated negligible specific binding to microsomal membrane fractions of tilapia kidney, liver, gill, or muscle. Tilapia PRL did not inhibit the binding of 125I-labeled ovine PRL to lactating rat liver membranes. The specific binding of 125I-labeled tilapia PRL to tilapia kidney membranes was greater with freshwater-acclimated than with seawater-acclimated fish suggesting that the tilapia kidney PRL-binding site is subject to physiological regulation.

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant ( K d) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity ( K d ≈ 6.6 · 10 −8 M). At high lectin concentrations, low affinity ( K d ≈ 6.7 · 10 −5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone. Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl- D-mannopyranoside.

Prolactin receptors in primary cultures of carcinogen-induced rat-mammary tumors

7,12-Dimethylbenz [ a] anthracene-induced rat mammary tumors were dissociated with collagenase and hyaluronidase and placed into primary culture. In most cultures, specific binding of 125I-labeled ovine prolactin was (i) lower than that for the original tumors unless bovine prolactin (1 μg/ml) had been added to the dissociation medium, and (ii) varied with the type of growth medium used. The level of prolactin binding in cultured cells was relatively constant for the first 7–10 days. Prolactin binding in cultured cell homogenates was maximal at pH 7.0, proportional to cell protein, specific for prolactin, and reached a steady state by 12 h at 22° C. The half-maximum inhibition of 125I-labeled prolactin binding by unlabeled prolactin was 100 ng/ml for cells grown in 5–1000 ng of prolactin/ml. After prolactin was removed from the growth medium, the level of available binding sites progressively increased, reached a maximum at 48 h and then declined. At 48 h, the dissociation constant for prolactin binding ( K d ∼ 1 × 10 −10 M) was comparable to that in tumors. In some cultured tumors, a 48-h treatment with 0.5 or 1.0 ng of prolactin/ml caused an apparent increase in the level of prolactin binding. Prolactin increased DNA synthesis and its removal caused a reduction in [ 3H]estradiol and [ 3H]R5020 binding to cultured cell cytosols.

ONTOGENY AND CONTROL OF PROLACTIN RECEPTORS IN THE MAMMARY GLAND AND LIVER OF VIRGIN, PREGNANT AND LACTATING RATS

Prolactin receptors were identified and partially characterized in the mammary gland of the rat. The binding of 125I-labelled ovine prolactin to a subcellular particulate fraction of rat mammary gland decreased between days 30 and 100 of age. Over the same period, binding to the liver increased and there was a significant negative correlation between prolactin binding in the two tissues. Binding to the mammary gland was low during pregnancy, increased in early lactation and declined after the litters were weaned. Binding to the liver was lower during lactation than during pregnancy or the period after weaning suggesting that tissue-specific factors may operate in the control of this receptor. In virgin rats, prolactin binding by the mammary gland was increased by oestrogen. This effect was blocked by hypophysectomy and partially restored by replacement therapy with prolactin. Hypothyroidism and treatment with progesterone also reduced the response to oestrogen. The maintenance of prolactin binding by the mammary gland of lactating rats depends on the presence of the ovaries and pituitary, thyroid and adrenal glands. Examination of the ratio epithelium: stroma suggests that prolactin acts by increasing the number of epithelial cells in the mammary gland and that thyroid, adrenal and ovarian hormones modulate the number of receptors per cell.