Characterization of a novel and functional human prolactin receptor isoform (deltaS1PRLr) containing only one extracellular fibronectin-like domain

Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71-373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the deltaS1 PRLr. Northern analysis revealed variable deltaS1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with deltaS1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the deltaS1 isoform bound ligand at a lower affinity than wild-type receptor. The deltaS1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.     

Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras

The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.     

Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications

Prolactin (PRL) contributes to the growth of normal and malignant breast tissues. PRL initiates signaling by engaging the PRL receptor (PRLr), a transmembrane (TM) receptor belonging to the cytokine receptor family. The accepted view has been that PRL activates the PRLr by inducing dimerization of the receptor, but recent reports show ligand-independent dimerization of other cytokine receptors. Using coimmunoprecipitation assays, we have confirmed ligand-independent dimerization of the PRLr in T47D breast cancer and HepG2 liver carcinoma cells. In addition, mammalian cells transfected with differentially epitope-tagged isoforms of the PRLr indicated that long, intermediate, and DeltaS1 PRLrs dimerized in a ligand-independent manner. To determine the domain(s) involved in PRLr ligand-independent dimerization, we generated PRLr constructs as follows: (1) the TM-ICD, which consisted of the TM domain and the intracellular domain (ICD) but lacked the extracellular domain (ECD), and (2) the ECD-TM, which consisted of the TM domain and the ECD but lacked the ICD. These constructs dimerized in a ligand-independent manner in mammalian cells, implicating a significant role for the TM domain in this process. These truncated PRLrs were functionally inert alone or in combination in cells lacking the PRLr. However, when introduced into cells containing endogenous PRLr, the ECD-TM inhibited human PRLr signaling, whereas the TM-ICD potentiated human PRLr signaling. These studies indicate that the ECD-TM and the TM-ICD are capable of modulating PRLr function. We also demonstrated an endogenous TM-ICD in T47D cells, suggesting that these findings are relevant to PRL-signaling pathways in breast cancer.     

Prolactin upregulates its receptors and inhibits lipolysis and leptin release in male rat adipose tissue

Prolactin (PRL) is recognized as a metabolic regulator during lactation, but little information exists on its actions in male adipose tissue. We examined whether PRL affects the expression of its receptors (PRLR), lipolysis, and adipokine secretion in male rats. Both long and short PRLR isoforms were induced 40-50-fold during differentiation of epididymal preadipocytes, with a 10-fold higher expression of the long isoform. PRL upregulated both isoforms before and after differentiation. PRL suppressed lipolysis in epididymal explants and mature adipocytes in a dose- and time-dependent manner, which was reversed by a Jak2 inhibitor. PRL also inhibited leptin, but not adiponectin, release. We conclude that PRL inhibits lipolysis and leptin release by acting directly on adipocytes via interaction with either of its receptors and activation of a Jak2-dependent signaling pathway(s). This is the first demonstration of substantial effects of PRL on male adipocytes.     

Quantification of PRL/Stat5 signaling with a novel pGL4-CISH reporter

Background  

Elevations of serum prolactin (PRL) are associated with an increased risk for breast cancer. PRL signaling through its prolactin receptor (PRLr) involves the Jak2/Stat5 pathway. Luciferase-based reporter assays have been widely used to evaluate the activity of this pathway. However, the existing reporters are often not sensitive enough to monitor the effect of PRL in this pathway.     

Prolactin inhibits activity of pyruvate kinase M2 to stimulate cell proliferation

Mitogenic and prosurvival effects underlie the tumorigenic roles of prolactin (PRL) in the pathogenesis of breast cancer. PRL signaling is mediated through its receptor (PRLr). A proteomics screen identified the pyruvate kinase M2 (PKM2), a glycolytic enzyme known to play an important role in tumorigenesis, as a protein that constitutively interacts with PRLr. Treatment of cells with PRL inhibited pyruvate kinase activity and increased the lactate content in human cells in a manner that was dependent on the abundance of PRLr, activation of Janus kinase 2, and tyrosine phosphorylation of the intracellular domain of PRLr. Knockdown of PKM2 attenuated PRL-stimulated cell proliferation. The extent of this proliferation was rescued by the knock-in of the wild-type PKM2 but not of its mutant insensitive to PRL-mediated inhibition. We discuss a hypothesis that the inhibition of PKM2 by PRL contributes to the PRL-stimulated cell proliferation.     

Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.     

Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.     

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src?K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src^?/? mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.     

Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.     

Different biological effects of unmodified prolactin and a molecular mimic of phosphorylated prolactin involve different signaling pathways

Previous work has shown that naturally phosphorylated prolactin antagonizes the growth-promoting activities of unmodified prolactin (U-PRL) and that this effect is duplicated by a molecular mimic, S179D PRL. At the same time, the S179D PRL is a superagonist with regard to expression of some PRL-regulated genes. We have asked whether the different activities of U-PRL and S179D PRL are the result of differential signaling. HC11 cells (a normal mouse mammary cell line) were grown to confluence, primed with hydrocortisone, and then exposed to the PRLs. A 15 min incubation of PRL-naive cells led to substantial tyrosine phosphorylation of Jak 2 and Stat 5a by U-PRL and an essentially equivalent Jak 2 activation by S179D PRL. The latter, however, was accompanied by reduced tyrosine phosphorylation of Stat 5a. EMSA analysis using a Stat 5 binding site showed both PRLs to cause equivalent binding of nuclear proteins and that most of what bound was complexed through Stat 5a. Phosphoamino acid analysis of Stat 5 showed S179D PRL to double the amount of serine phosphorylation versus that seen with U-PRL. Analysis of the MAP kinase pathway showed U-PRL capable of activation of ERKs 1 and 2 but that signaling via ERKs 1 and 2 was greater with S179D PRL. A 7-day incubation in either PRL increased beta-casein mRNA levels, but S179D PRL caused a 2-fold increase over that seen with U-PRL. The increase, over that seen with U-PRL, was blocked by the MAP kinase inhibitor, PD98059. After 7 days of treatment with S179D PRL, expression of the short PRL receptor was doubled, and signaling showed a greater dependence on the MAP kinase pathway (2.9-fold increase in ERK 1 and 2 activation). We conclude that although both PRLs use both pathways to some extent, U-PRL signals primarily through Jak 2-Stat 5 whereas S179D PRL signals primarily through the MAP kinase pathway especially after prolonged exposure. This is the first demonstration of differential involvement of signaling pathways by different forms of PRL.     

A catalytically active Jak2 is required for the angiotensin II-dependent activation of Fyn.

Recent work with interleukins has shown a convergence of tyrosine phosphorylation signal transduction cascades at the level of the Janus and Src families of tyrosine kinases. Here we demonstrate that activation of the seven-transmembrane AT(1) receptor by angiotensin II induces a physical association between Jak2 and Fyn, in vivo. This association requires the catalytic activity of Jak2 but not Fyn. Deletion studies indicate that the region of Jak2 that binds Fyn is located between amino acids 1 and 240. Studies of the Fyn SH2 and SH3 domains demonstrate that the SH2 domain plays the primary role in Jak2/Fyn association. Not surprisingly, this domain shows a marked preference for tyrosine-phosphorylated Jak2. Surface plasmon resonance estimated the dissociation equilibrium constant (K(d)) of this association to be 2.36 nM. Last, in vivo studies in vascular smooth muscle cells show that, in response to angiotensin II, Jak2 activation is required for Fyn activation and induction of the c-fos gene. The significance of these data is that Jak2, in addition to serving as a critical angiotensin II activated signal transduction kinase, also functions as a docking protein and participates in the activation of Fyn by providing phosphotyrosine residues that bind the SH2 domain of Fyn.     

The c-Kit/D816V mutation eliminates the differences in signal transduction and biological responses between two isoforms of c-Kit

Activating mutations of codon 816 of the Kit gene have been implicated in malignant cell growth of acute myeloid leukemia (AML), systemic mastocytosis and germ cell tumors. Substitution of aspartic acid with valine (D816V) renders the receptor independent of ligand for activation and signaling. Wild-type c-Kit is a tyrosine kinase receptor that requires its ligand, stem cell factor (SCF), for activation. Several isoforms of c-Kit exist as a result of alternative mRNA splicing, of which two are characterized by the presence or absence of four amino acids (GNNK? and GNNK+, respectively) in the extracellular domain. The two isoforms show differences in signal transduction and biological activities and the shorter isoform seems to be highly expressed than the longer isoform in human malignancies. In this study we analysed the signal transduction downstream of the oncogenic c-Kit mutant D816V in an isoform specific context, using the hematopoietic cell line Ba/F3 stably transfected with the different versions of isoform and mutant receptor. Our data show that in contrast to the differences shown in the activation of wild-type c-Kit isoforms, both isoforms of c-Kit/D816V are constitutively phosphorylated to the same extent. By the use of Western blot analysis we investigated the activation of different signaling proteins and found that both D816V/GNNK? and D816V/GNNK+ constitutively phosphorylated Gab2, Shc, SHP-2 and Cbl to almost the same extent as c-Kit/GNNK?. In addition, both isoforms of c-Kit/D816V induced SCF-independent cell survival and proliferation equally well. This is in contrast to wild-type c-Kit, where c-Kit/GNNK? induced better cell survival and stronger proliferation than c-Kit/GNNK+, and both required stimulation with SCF. Taken together, these findings reveal that the differences in downstream signal transduction and biological responses between the two GNNK isoforms are eliminated by the D816V mutant.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Regulation of Jak kinases by intracellular leptin receptor sequences

Leptin signals the status of body energy stores via the leptin receptor (LR), a member of the Type I cytokine receptor family. Type I cytokine receptors mediate intracellular signaling via the activation of associated Jak family tyrosine kinases. Although their COOH-terminal sequences vary, alternatively spliced LR isoforms (LRa-LRd) share common NH(2)-terminal sequences, including the first 29 intracellular amino acids. The so-called long form LR (LRb) activates Jak-dependent signaling and is required for the physiologic actions of leptin. In this study, we have analyzed Jak activation by intracellular LR sequences under the control of the extracellular erythropoeitin (Epo) (Epo receptor/LRb chimeras). We show that Jak2 is the requisite Jak kinase for signaling by the LRb intracellular domain and confirm the requirement for the Box 1 motif for Jak2 activation. A minimal LRb intracellular domain for Jak2 activation includes intracellular amino acids 31-48. Although the sequence requirements for intracellular amino acids 37-48 are flexible, intracellular amino acids 31-36 of LRb play a critical role in Jak2 activation and contain a loose homology motif found in other Jak2-activating cytokine receptors. The failure of short form sequences to function in Jak2 activation reflects the absence of this motif.     

Differences in prolactin receptor (PRLR) in mouse and human fallopian tubes: evidence for multiple regulatory mechanisms controlling PRLR isoform expression in mice

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.     

Multiple internalization motifs differentially used by prolactin receptor isoforms mediate similar endocytic pathways

Prolactin (PRL) regulates a variety of physiological processes, including mammary gland growth and differentiation, modulation of behavior, and immune function. A long PRL receptor (lPRLR) and short (sPRLR) isoform were identified in ruminants and rodents, which differ in their distal cytoplasmic domains and possess markedly distinct signaling capacities. Here we compared endocytosis of the bovine isoforms and found that the lPRLR internalized faster than the sPRLR, which would contribute to short-term down-regulation of lPRLR signaling at targets expressing both isoforms. Multiple motifs were required to mediate internalization of the lPRLR, including a phenylalanine (F290) plus a nearby dileucine, and three dileucines proximal to amino acid 272. This is different from the closely related GH receptor that requires only the phenyl-alanine-containing motif for endocytosis. Truncated lPRLR (cT272), which is the same length as the sPRLR and contained the proximal three dileucines, internalized at the same rate as the full-length lPRLR. Finally, the two dileucines shared by the sPRLR were able to mediate similar endocytic pathways as the lPRLR, as revealed by overexpression of mutant dynamin and clathrin hub, despite the slower rate. These studies define the basis of cellular trafficking of PRLR isoforms and increase our understanding of control of target cell responsiveness by PRL.