Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.

One of the potential biomarkers for ovarian cancer patients is high serum level of prolactin (PRL), which is a growth factor that may promote tumor cell growth. The prolactin receptor (PRLR) and human cytomegalovirus (HCMV) proteins are frequently detected in ovarian tumor tissue specimens, but the potential impact of HCMV infection on the PRL system have so far not been investigated. In this study, HCMV's effects on PRL and PRLR expression were assessed in infected ovarian cancer cells (SKOV3) by PCR and Western blot techniques. The levels of both PRL and PRLR transcripts as well as the corresponding proteins were highly increased in HCMV-infected SKOV3 cells. Tissue specimens obtained from 10 patients with ovarian cancer demonstrated high expression of PRLR, HCMV-IE, and pp65 proteins. Extensive expression of PRLR was detected in all examined ovarian tumor tissue specimens except for one from a patient who had focal expression of PRLR and this patient was HCMV-negative in her tumor. In conclusion, PRL and PRLR were induced to high levels in HCMV-infected ovarian cancer cells and PRLR expression was extensively detected in HCMV-infected ovarian tissue specimens. Highly induced PRL and PRLR by HCMV infection may be of relevance for the oncomodulatory role of this virus in ovarian cancer.

Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGβ gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.

Kisspeptin (Kp) regulates prolactin (PRL) in an estradiol-dependent manner. We investigated the interaction between ovarian steroid receptors and Kp in the control of PRL secretion. Intracerebroventricular injections of Kp-10 or Kp-234 were performed in ovariectomized (OVX) rats under different hormonal treatments. Kp-10 increased PRL release and decreased 3,4-dihydroxyphenylacetic acid levels in the median eminence (ME) of OVX rats treated with estradiol (OVX+E), which was prevented by tamoxifen. Whereas these effects of Kp-10 were absent in OVX rats, they were replicated in OVX rats treated with selective agonist of estrogen receptor (ER)α, propylpyrazole triol, but not of ERβ, diarylpropionitrile. Furthermore, the Kp-10-induced increase in PRL was two times higher in OVX+E rats also treated with progesterone (OVX+EP), which was associated with a reduced expression of both tyrosine hydroxylase (TH) and Ser40-phosphorylated TH in the ME. Kp-10 also reduced dopamine levels in the ME of OVX+EP rats, an effect blocked by the progesterone receptor (PR) antagonist RU486. We also determined the effect of Kp antagonism with Kp-234 on the estradiol-induced surges of PRL and luteinizing hormone (LH), using tail-tip blood sampling combined with ultrasensitive enzyme-linked immunosorbent assay. Kp-234 impaired the early phase of the PRL surge and prevented the LH surge in OVX+E rats. Thus, we provide evidence that Kp stimulation of PRL release requires ERα and is potentiated by progesterone via PR activation. Moreover, alongside its essential role in the LH surge, Kp seems to play a role in the peak phase of the estradiol-induced PRL surge.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the 'islet pregnancy gene signature', which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells.

Prolactin (PRL) is elevated in B-cell-mediated lymphoproliferative diseases and promotes B-cell survival. Whether PRL or PRL receptors drive the evolution of B-cell malignancies is unknown. We measure changes in B cells after knocking down the pro-proliferative, anti-apoptotic long isoform of the PRL receptor (LFPRLR) in vivo in systemic lupus erythematosus (SLE)- and B-cell lymphoma-prone mouse models, and the long plus intermediate isoforms (LF/IFPRLR) in human B-cell malignancies. To knockdown LF/IFPRLRs without suppressing expression of the counteractive short PRLR isoforms (SFPRLRs), we employ splice-modulating DNA oligomers. In SLE-prone mice, LFPRLR knockdown reduces numbers and proliferation of pathogenic B-cell subsets and lowers the risk of B-cell transformation by downregulating expression of activation-induced cytidine deaminase. LFPRLR knockdown in lymphoma-prone mice reduces B-cell numbers and their expression of BCL2 and TCL1. In overt human B-cell malignancies, LF/IFPRLR knockdown reduces B-cell viability and their MYC and BCL2 expression. Unlike normal B cells, human B-cell malignancies secrete autocrine PRL and often express no SFPRLRs. Neutralization of secreted PRL reduces the viability of B-cell malignancies. Knockdown of LF/IFPRLR reduces the growth of human B-cell malignancies in vitro and in vivo. Thus, LF/IFPRLR knockdown is a highly specific approach to block the evolution of B-cell neoplasms.

The receptors for the pituitary hormones growth hormone (GH), prolactin (PRL) and somatolactin (SL), and the hematopoietic hormones erythropoietin (EPO) and thrombopoietin (TPO), comprise a structurally related family in the superfamily of cytokine class-I receptors. GH, PRL and SL receptors have a wide variety of effects in development, osmoregulation, metabolism and stimulation of growth, while EPO and TPO receptors guide the production and differentiation of erythrocytes and thrombocytes, respectively. The evolution of the receptors for GH, PRL and SL has been partially investigated by previous reports suggesting different time points for the hormone and receptor gene duplications. This raises questions about how hormone-receptor partnerships have emerged and evolved. Therefore, we have investigated in detail the expansion of this receptor family, especially in relation to the basal vertebrate (1R, 2R) and teleost (3R) tetraploidizations. Receptor family genes were identified in a broad range of vertebrate genomes and investigated using a combination of sequence-based phylogenetic analyses and comparative genomic analyses of synteny. We found that 1R most likely generated EPOR/TPOR and GHR/PRLR ancestors; following this, 2R resulted in EPOR and TPOR genes. No GHR/PRLR duplicate seems to have survived after 2R. Instead the single GHR/PRLR underwent a local duplication sometime after 2R, generating separate syntenic genes for GHR and PRLR. Subsequently, 3R duplicated the gene pair in teleosts, resulting in two GHR and two PRLR genes, but no EPOR or TPOR duplicates. These analyses help illuminate the evolution of the regulatory mechanisms for somatic growth, metabolism, osmoregulation and hematopoiesis in vertebrates.

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth.

The abilities of dopamine (DA) and the partial DA D2 receptor agonist (-)-(3-hydroxyphenyl)-N-n-propylpiperidine, (-)-3-PPP, to suppress prolactin (PRL) release induced by any of five different PRL secretagogues in GH4C1 cells transfected with the human D2 receptor (short isoform) were investigated. Whereas DA reduced the response to all five secretagogues. (-)-3-PPP reduced the response to vasoactive intestinal peptide (VIP) and thyrotropin-releasing hormone (TRH), but not to high medium potassium (K+) or to the potassium channel antagonist tetraethylammonium (TEA). (-)-3-PPP tended to reduce the PRL release induced by the Ca2+ channel agonist BAY K-8644 (BAY); however, this effect of the partial agonist was modest and not significant. Whereas the effects of both DA and (-)-3-PPP on the PRL response to VIP and TRH were counteracted by co-incubation with the D2 antagonist raclopride, the effects of DA on the PRL response to K+, BAY, and TEA were antagonized by co-incubation with either raclopride or (-)-3-PPP. The results show that, at a given receptor density, the intrinsic activity of a partial D2 agonist with respect to D2-mediated suppression of PRL release may vary from agonism to antagonism depending on which intracellular transduction systems that are being concomitantly activated.

Post-parturient behavior of mammalian females is essential for early parent-offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

Growth hormone receptor (GHR) and prolactin receptor (PRLR) in jawed vertebrates were thought to arise after the divergence of gnathostomes from a basal vertebrate. In this study we have identified two genes encoding putative GHR and PRLR in sea lamprey (Petromyzon marinus) and Arctic lamprey (Lethenteron camtschaticum), extant members of one of the oldest vertebrate groups, agnathans. Phylogenetic analysis revealed that lamprey GHR and PRLR cluster at the base of gnathostome GHR and PRLR clades, respectively. This indicates that distinct GHR and PRLR arose prior to the emergence of the lamprey branch of agnathans. In the sea lamprey, GHR and PRLR displayed a differential but overlapping pattern of expression; GHR had high expression in liver and heart tissues, whereas PRLR was expressed highly in the brain and moderately in osmoregulatory tissues. Branchial PRLR mRNA levels were significantly elevated by stage 5 of metamorphosis and remained elevated through stage 7, whereas levels of GHR mRNA were only elevated in the final stage (7). Branchial expression of GHR increased following seawater (SW) exposure of juveniles, but expression of PRLR was not significantly altered. The results indicate that GHR and PRLR may both participate in metamorphosis and that GHR may mediate SW acclimation.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

The epithelial-mesenchymal transition (EMT) process is known to play an essential role in tumor progression, metastasis and resistance to therapy. This report evaluated the prognostic value of co-expression of the receptor for prolactin (PRLR), a suppressor of EMT, and the receptors for transforming growth factor β (TGFβRI and TGFβRII), an inducer of EMT, in association with different clinicopathological parameters using TMA of 102 breast cancer patients and publicly available data on breast cancer patients. Interestingly, the results revealed that malignant tissues had significantly lower levels of concomitant protein expression of these receptors in comparison to normal/benign breast tissue. In addition, a higher level of concomitant expression was also observed in less aggressive breast cancer phenotypes, including low grade tumors, luminal breast cancer subtype, and less advanced stages of the disease (lymph node negative and early stages). Moreover, the results also showed that the expression of a gene signature composed of PRLR/TGFβRI/TGFβRII correlates more with differentiated grade I tumors, and identified a subset of patients showing better survival outcomes evident in luminal B and HER-2 enriched molecular subtypes. Together, these results indicate that loss of the co-expression of PRLR, TGFβRI and TGFβRII is indicative of aggressiveness and poor patient survival outcomes in breast cancer.

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.

Neoplasms of the mammary gland are among the most common diseases in female domestic dogs (Canis familiaris). It is assumed that reproductive hormones influence tumorigenesis in this species, although the precise role of the endocrine milieu and reproductive state is subject to continuing discussion. In line with this, a recent systematic review of available data on the development of mammary neoplasms revealed weak evidence for risk reduction after neutering and an effect of age at neutering. Investigation of several hormone receptors has revealed decreased expression of estrogen receptor-alpha (ERα, ESR1), progesterone (P4) receptor (PGR), prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) associated with neoplastic differentiation of mammary tissues. In other studies, increased levels of estrogens, progesterone and prolactin were found in serum and/or tissue homogenates of dogs with malignant neoplasms. However, the association between these entities within one animal population was never previously examined. Therefore, this study investigated the association between circulating serum concentrations of estradiol-17β, progesterone and prolactin, and gene expression of ERα (ESR1), ERβ (ESR2), PGR, PRLR, PRL and GHR, with respect to reproductive state (spayed vs. intact) and cycle stage (anestrus vs. diestrus). Additionally, the expression of E-cadherin (CDH-1) was evaluated as a possible indicator of metastatic potential.

Cervical cancer (CC) is the second most common cancer in less developed countries and the second leading cause of death by cancer in women worldwide. The 99% of CC patients are infected with the Human Papilloma Virus (HPV), being HPV16 and HPV18 infection the most frequent. Even though HPV is considered to be a necessary factor for the development of CC, it is not enough, as it requires the participation of other factors such as the hormonal ones. Several studies have demonstrated the requirement of estrogen and its receptors (ERα, ERβ, and GPER) in the precursor lesions progress towards CC. Also, prolactin (PRL) and its receptor (PRLR) have been associated with CC. The molecular mechanisms underlying the cooperation of these hormones with the viral oncoproteins are not well elucidated. For this reason, this study focused on analyzing the contribution of 17β-estradiol (E2), PRL, and HPV on the expression and localization of hormone receptors, as well as to evaluate whether these hormones may promote greater expression of HPV oncogenes and contribute to tumor progression.

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm11-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm11-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm11-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y1, Y2 and Y5) receptors. Palm11-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.

In this study, structural analysis of grass carp prolactin (PRL) gene was performed and the signaling mechanisms for pituitary adenylate cyclase-activating peptide (PACAP) regulation of PRL promoter activity were investigated. In αT3-1 cells, PRL promoter activity could be induced by oPACAP38 which was blocked by PACAP antagonist but not the VIP antagonist. The stimulatory effect of oPACAP38 was mimicked by activation of AC/cAMP and voltage-sensitive Ca2+ channel (VSCC) signaling, or induction of Ca2+ entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca2+, VSCC blockade, calmodulin (CaM) antagonism, and inactivation of CaM kinase II. Similar sensitivity to L-type VSCC, CaM and CaM kinase II inhibition were also observed by substituting cAMP analog for oPACAP38 as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca2+]i immobilization via IP3 receptors, and blockade of PI3K/P70S6K pathway. The PACAP-induced PRL promoter activation may involve transactivation of the transcription factor CREB. These results suggest that PACAP can stimulate PRL promoter activation by PAC1 mediated functional coupling of the Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P70S6K cascades, may also be involved in PACAP induction of PRL gene transcription.

We present seven new vertebrate homologs of the prolactin-releasing hormone receptor (PRLHR) and show that these are found as two separate subtypes, PRLHR1 and PRLHR2. Analysis of a number of vertebrate sequences using phylogeny, pharmacology, and paralogon analysis indicates that the PRLHRs are likely to share a common ancestry with the neuropeptide Y (NPY) receptors. Moreover, a micromolar level of NPY was able to bind and inhibit completely the PRLH-evoked response in PRLHR1-expressing cells. We suggest that an ancestral PRLH peptide started coevolving with a redundant NPY binding receptor, which then became PRLHR, approximately 500 million years ago. The PRLHR1 subtype was shown to have a relatively high evolutionary rate compared to receptors with fixed peptide preference, which could indicate a drastic change in binding preference, thus supporting this hypothesis. This report suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions.

Dopamine (DA) D2-like (and D1-like) receptors are suggested to mediate the dopamine actions in the anterior pituitary and/or CNS of birds. However, the information regarding the structure, functionality, and expression of avian D2-like receptors have not been fully characterized. In this study, we cloned two D2-like receptors (cDRD2, cDRD4) from chicken brain using RACE PCR. The cloned cDRD4 is a 378-amino acid receptor, which shows 57% amino acid (a.a.) identity with mouse DRD4. As in mammals, two cDRD2 isoforms, cDRD2L (long isoform, 437 a.a.) and cDRD2S (short isoform, 408 a.a.), which differ in their third intracellular loop, were identified in chickens. Using cell-based luciferase reporter assays or Western blot, we demonstrated that cDRD4, cDRD2L and cDRD2S could be activated by dopamine and quinpirole (a D2-like receptor agonist) dose-dependently, and their activation inhibits cAMP signaling pathway and stimulates MAPK/ERK signaling cascade, indicating that they are functional receptors capable of mediating dopamine actions. Quantitative real-time PCR revealed that cDRD2 and cDRD4 are widely expressed in chicken tissues with abundant expression noted in anterior pituitary, and their expressions are likely controlled by their promoters near exon 1, as demonstrated by dual-luciferase reporter assays in DF-1 cells. In accordance with cDRD2/cDRD4 expression in the pituitary, DA or quinpirole could partially inhibit vasoactive intestinal peptide-induced prolactin expression in cultured chick pituitary cells. Together, our data proves the functionality of DRD2 and DRD4 in birds and aids to uncover the conserved roles of DA/D2-like receptor system in vertebrates, such as its action on the pituitary.