Multimeric growth hormone receptor complexes serve as signaling platforms.

Growth hormone (GH) signaling is required for promoting longitudinal body growth, stem cell activation, differentiation, and survival and for regulation of metabolism. Failure to adequately regulate GH signaling leads to disease: excessive GH signaling has been connected to cancer, and GH insensitivity has been reported in cachexia patients. Since its discovery in 1989, the receptor has served a pivotal role as the prototype cytokine receptor both structurally and functionally. Phosphorylation and ubiquitylation regulate the GH receptor (GHR) at the cell surface: two ubiquitin ligases (SCF(ßTrCP2) and CHIP) determine the GH responsiveness of cells by controlling its endocytosis, whereas JAK2 initiates the JAK/STAT pathway. We used blue native electrophoresis to identify phosphorylated and ubiquitylated receptor intermediates. We show that GHRs occur as ∼500-kDa complexes that dimerize into active ∼900-kDa complexes upon GH binding. The dimerized complexes act as platforms for transient interaction with JAK2 and ubiquitin ligases. If GH and receptors are made in the same cell (autocrine mode), only limited numbers of ∼900-kDa complexes are formed. The experiments reveal the dynamic changes in post-translational modifications during GH-induced signaling events and show that relatively simple cytokine receptors like GHRs are able to form higher order protein complexes. Insight in the complex formation of cytokine receptors is crucially important for engineering cytokines that control ligand-induced cell responses and for generating a new class of therapeutic agents for a wide range of diseases.

SUMOylation is a regulator of the translocation of Jak2 between nucleus and cytosol.

Jak2 (Janus kinase 2) initiates the signal transduction of many cytokine receptors. We discovered that Jak2 is SUMOylated on multiple lysine residues by SUMO2/3 (small ubiquitin-related modifier 2/3) chains. Analysis of Jak2 mutants revealed that Jak2 SUMOylation depends on the presence of an active catalytic site. We used the GH (growth hormone) receptor to study the physiological relevance of Jak2 SUMOylation. Both GH stimulation and several other environmental stressors increased Jak2 SUMOylation. Cell fractionation showed that SUMOylated Jak2 is mainly present in the nucleus. The constitutively active V617F Jak2 mutant, implicated in myeloproliferative diseases, was highly SUMOylated in the absence of stimuli. These data provide evidence that Jak2 SUMOylation controls Jak2 shuttling between cytoplasm and nucleus.

ßTrCP interacts with the ubiquitin-dependent endocytosis motif of the GH receptor in an unconventional manner.

GH (growth hormone) binding to the GHR (GH receptor) triggers essential signalling pathways that promote growth and metabolic regulation. The sensitivity of the cells to GH is mainly controlled by the endocytosis of the receptor via ßTrCP (ß-transducin repeat-containing protein). In the present study, we show that ßTrCP interacts directly via its WD40 domain with the UbE (ubiquitin-dependent endocytosis) motif in GHR, promoting GHR ubiquitination in vitro. NMR experiments demonstrated that the UbE motif is essentially unstructured, and, together with functional mapping of the UbE and ßTrCP WD40 residues necessary for binding, led to a unique interaction model of ßTrCP with GHR-UbE. This interaction is different from the conventional ßTrCP-substrate interactions described to date. This interaction therefore represents a promising specific target to develop drugs that inhibit GHR endocytosis and increase GH sensitivity in cachexia patients.

Ubc13 and COOH terminus of Hsp70-interacting protein (CHIP) are required for growth hormone receptor endocytosis.

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(ßTrCP) (Skp Cullin F-box). Despite a specific interaction between ß-transducin repeat-containing protein (ßTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(ßTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to ßTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.

WIP1 is a novel specific target for growth hormone action.

DNA damage repair (DDR) is mediated by phosphorylating effectors ATM kinase, CHK2, p53, and γH2AX. We showed earlier that GH suppresses DDR by suppressing pATM, resulting in DNA damage accumulation. Here, we show GH acting through GH receptor (GHR) inducing wild-type p53-inducible phosphatase 1 (WIP1), which dephosphorylated ATM and its effectors in normal human colon cells and three-dimensional human intestinal organoids. Mice bearing GH-secreting xenografts exhibited induced colon WIP1 with suppressed pATM and γH2AX. WIP1 was also induced in buffy coats derived from patients with elevated GH from somatotroph adenomas. In contrast, decreased colon WIP1 was observed in GHR-/- mice. WIP1 inhibition restored ATM phosphorylation and reversed GH-induced DNA damage. We elucidated a novel GH signaling pathway activating Src/AMPK to trigger HIPK2 nuclear-cytoplasmic relocation and suppressing WIP1 ubiquitination. Concordantly, blocking either AMPK or Src abolished GH-induced WIP1. We identify WIP1 as a specific target for GH-mediated epithelial DNA damage accumulation.

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance.

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthetase (FPGS), which normally promotes intracellular accumulation and activity of natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. Here, we describe a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase and downstream one-carbon metabolism. Contrary to methotrexate, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folates for interaction with dihydrofolate reductase. We show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1, whereas FPGS-high CRC organoids are more sensitive to methotrexate. Our results argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, using a vulnerability created by polyglutamylation deficiency.

SCF(TrCP) acts in endosomal sorting of the GH receptor.

The ubiquitin ligase SCF(TrCP) is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF(TrCP) is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF(TrCP) is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.

Small molecules to regulate the GH/IGF1 axis by inhibiting the growth hormone receptor synthesis.

Growth hormone (GH) and insulin-like growth factor-1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10-30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA-MB-231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule.

Growth Hormone Receptor Regulation in Cancer and Chronic Diseases.

The GHR signaling pathway plays important roles in growth, metabolism, cell cycle control, immunity, homeostatic processes, and chemoresistance via both the JAK/STAT and the SRC pathways. Dysregulation of GHR signaling is associated with various diseases and chronic conditions such as acromegaly, cancer, aging, metabolic disease, fibroses, inflammation and autoimmunity. Numerous studies entailing the GHR signaling pathway have been conducted for various cancers. Diverse factors mediate the up- or down-regulation of GHR signaling through post-translational modifications. Of the numerous modifications, ubiquitination and deubiquitination are prominent events. Ubiquitination by E3 ligase attaches ubiquitins to target proteins and induces proteasomal degradation or starts the sequence of events that leads to endocytosis and lysosomal degradation. In this review, we discuss the role of first line effectors that act directly on the GHR at the cell surface including ADAM17, JAK2, SRC family member Lyn, Ubc13/CHIP, proteasome, ßTrCP, CK2, STAT5b, and SOCS2. Activity of all, except JAK2, Lyn and STAT5b, counteract GHR signaling. Loss of their function increases the GH-induced signaling in favor of aging and certain chronic diseases, exemplified by increased lung cancer risk in case of a mutation in the SOCS2-GHR interaction site. Insight in their roles in GHR signaling can be applied for cancer and other therapeutic strategies.

Hypoxia-mediated degradation of Na,K-ATPase via mitochondrial reactive oxygen species and the ubiquitin-conjugating system.

We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes Na,K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O2 had a decrease in Na,K-ATPase activity and oxygen consumption. The total cell pool of alpha1 Na,K-ATPase protein decreased on exposure to 1.5% O2 for 30 hours, whereas the plasma membrane Na,K-ATPase was 50% degraded after 2 hours of hypoxia, which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40 degrees C and 1.5% O2, the degradation of the alpha1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient rho(0) cells and in cells transfected with small interfering RNA against Rieske iron sulfur protein, the hypoxia-mediated Na,K-ATPase degradation was prevented. The catalase/superoxide dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na,K-ATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na+ pump degradation. Accordingly, we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma membrane Na,K-ATPase via the ubiquitin-conjugating system.

Autocrine growth hormone: effects on growth hormone receptor trafficking and signaling.

GH and GH receptor are expressed in many extrapituitary tissues, permitting autocrine/paracrine activity. Autocrine GH has regulatory functions in embryonic development and cellular differentiation and proliferation and is reported to be involved in the development and metastasis of tumor cells. To understand the principles of transport and signaling of autocrine GH and GH receptor, we used a model system to express both proteins in the same cell. Our experiments show that GH binds the GH receptor immediately after synthesis in the endoplasmic reticulum and facilitates maturation of GH receptor. The hormone-receptor complexes arrive at the cell surface where exogenously added GH is unable to bind these receptors. Autocrine GH activates the GH receptors, but signal transduction occurs only after exiting the endoplasmic reticulum. This model study explains why autocrine GH-producing cells may be insensitive for GH (antagonist) treatment and clarifies autocrine signaling events.

Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes.

In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron microscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.

Proteasome regulates the delivery of LDL receptor-related protein into the degradation pathway.

The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin-proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway.

Fos-Zippered GH Receptor Cytosolic Tails Act as Jak2 Substrates and Signal Transducers.

Members of the Janus kinase (Jak) family initiate the majority of downstream signaling events of the cytokine receptor family. The prevailing principle is that the receptors act in dimers: 2 Jak2 molecules bind to the cytosolic tails of a cytokine receptor family member and initiate Jak-signal transducer and activator of transcription signaling upon a conformational change in the receptor complex, induced by the cognate cytokine. Due to the complexity of signaling complexes, there is a strong need for in vitro model systems. To investigate the molecular details of the Jak2 interaction with the GH receptor (GHR), we used cytosolic tails provided with leucine zippers derived from c-Fos to mimic the dimerized state of GHR. Expressed together with Jak2, fos-zippered tails, but not unzippered tails, were stabilized. In addition, the Jak-signal transducer and activator of transcription signaling pathway was activated by the fos-zippered tails. The stabilization depended also on α-helix rotation of the zippers. Fos-zippered GHR tails and Jak2, both purified from baculovirus-infected insect cells, interacted via box1 with a binding affinity of approximately 40nM. As expected, the Jak kinase inhibitor Ruxolitinib inhibited the stabilization but did not affect the c-Fos-zippered GHR tail-Jak2 interaction. Analysis by blue-native gel electrophoresis revealed high molecular-weight complexes containing both Jak2 and nonphosphorylated GHR tails, whereas Jak2-dissociated tails were highly phosphorylated and monomeric, implying that Jak2 detaches from its substrate upon phosphorylation.

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.

Dimerization and signal transduction of the growth hormone receptor.

GH binding to cell surface-localized GH receptors (GHRs) induces a conformational change of the dimerized receptors, resulting in activation of Janus kinase 2 and downstream signaling pathways. Interactions between the extracellular subdomain 2 of adjacent GHR polypeptides result in a 500-A2 contact interface, which has previously been suggested to stabilize the GH-(GHR)2 complex. In this study, we investigated further the role of subdomain 2 in GHR function. Amino acids that participate in (e.g. aspartic acid 152, tyrosine 200, or serine 201) or lie close to (e.g. asparagine 143 or cysteine 241) the contact interface were mutated in rabbit GHR. Surprisingly, none of the mutations affected GHR dimerization, as demonstrated by coimmunoprecipitation of a truncated, epitope-tagged GHR. However, signal transduction of GHR(D152H), GHR(Y200D), and GHR(S201K) mutants was precluded. More insight into the molecular mechanism of the signaling defect was obtained when we examined the effect of the mutations on the integrity of the GH-(GHR)2 complex in a protease-protection assay. In contrast to wild-type GHR, GHR(N143K), and GHR(C241S), the GHR(D152H), GHR(Y200D), and GHR(S201K) mutants were not protected against protease digestion by GH, indicating that a structural change is prevented. Together, we provide new evidence for a critical role of aspartic acid 152, tyrosine 200, and serine 201 of the GHR contact interface in the GH-induced conformational change to a signaling-competent complex rather than in GHR dimerization.

The growth hormone receptor interacts with its sheddase, the tumour necrosis factor-alpha-converting enzyme (TACE).

Proteolysis of the GHR (growth hormone receptor) occurs at the cell surface and results in the release of its extracellular domain, the GHBP (growth hormone-binding protein). TACE (tumour necrosis factor-alpha-converting enzyme) has been identified as a putative protease responsible for GHR cleavage. However, GHR-TACE interaction has not been observed until now. Here, we identified TACE in Chinese hamster cells and confirmed processing and cell-surface expression. Interaction between GHR and TACE was only observed after growth hormone binding. As the growth hormone-GHR(2) complex is a poor substrate for TACE, we conclude that the GHR-TACE interaction precedes proteolysis, and is transient. Furthermore, we demonstrate that TACE is present in endosomes, where it partly co-localizes with endocytosed growth hormone ligand.

Automation of a phospho-STAT5 staining procedure for flow cytometry for application in drug discovery.

Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate this assay, to allow the direct screening of drugs targeting signaling pathways. We show here the sensitivity of this fully automated assay on human growth hormone (hGH)-driven JAK/STAT5-activated IM-9 cells, and we discuss the throughput of this system, which is compatible with medium-throughput drug screening. Because the kit works directly on whole blood samples, ex-vivo assays are also possible with this approach, which could allow for the screening of drugs under more physiological conditions.

The ubiquitin-proteasome pathway regulates the availability of the GH receptor.

GH promotes not only longitudinal growth in children but is active throughout life in protein, fat, and carbohydrate metabolism. The multiple actions of GH start when GH binds to the cell surface-expressed GH receptor. Effectiveness of the hormone depends both on its presence in the circulation and the availability of receptors at the cell surface of target cells. In this study, we examined the role of the ubiquitin-proteasome pathway in regulating GH receptor availability. We show that receptor turnover is rapid, and almost 3-fold prolonged in the internalization-deficient mutant GH receptor (F327A). Using a monovalent GH antagonist, B2036, we could quantify the internalization of the nonactivated receptor. By comparing internalization of the receptor with shedding of the GH-binding protein, we show that in Chinese hamster lung cell lines, internalization followed by lysosomal degradation is the major pathway for receptor degradation and that the ubiquitin-proteasome pathway controls this process. Inhibition of endocytosis resulted in a 200% increase in receptor availability at the cell surface at steady state.

Dimerization, ubiquitylation and endocytosis go together in growth hormone receptor function.

Internalization of membrane proteins has been studied for more than three decades without solving all the underlying mechanisms. Our knowledge of the clathrin-coated endocytosis is sufficient to understand the basic principles. However, more detailed insight is required to recognize why different proteins enter clathrin-coated pits with different rates and affinities. In addition to clathrin coat components, several adapter systems and even more accessory proteins have been described to preselect membrane proteins before they can enter cells. Recent experimental data have identified the ubiquitin-proteasome system as a regulatory system both in endocytic and lysosomal membrane traffic. This system is well-known for its basic regulatory function in protein degradation, and controls a magnitude of key events. In this review, we will discuss the complexity and implications of this mechanism for membrane trafficking with emphasis on the growth hormone receptor.