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Phosphorylation and Ubiquitination in Growth Hormone Receptor Endocytosis and Signalling Paranimfen: Johan Slotman Agnes Van Rossum Printer: Ponsen en Looijen, Wageningen The research described in this thesis was performed in the Department of Cell Biology at the University Medical Center in Utrecht, the Netherlands and was financially supported by the ‘Nederlandse Organisatie voor Wetenschappelijk Onderzoek, gebied aard- en levenswetenschappen (ALW) (NWO-814-02-011). Phosphorylation and ubiquitination in Growth Hormone Receptor endocytosis and signalling Fosforylering en ubiquitinering in groeihormoonreceptor endocytose en signalering (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 6 april 2010 des middags te 12.45 uur door Joyce Putters geboren op 8 september 1981 te Schelluinen Promotor: Prof. dr. G.J.A.M. Strous Printing of this thesis was financially supported by the ‘Nederlandse vereniging voor Groeihormoondeficiëntie en Groeihormoonbehandeling’. Contents Abbreviations 6 Chapter I Introduction 7 Chapter II The ubiquitin ligase SCF(βTrCP) regulates the 41 degradation of the growth hormone receptor Chapter III Jak2 is a negative regulator of ubiquitin-dependent 61 endocytosis of the growth hormone receptor Chapter IV Specificity, location and function of βTrCP isoforms 79 and splice variants Chapter V The contribution of tyrosine residues to growth 95 hormone receptor signalling Chapter VI Summarising discussion 109 Nederlandse samenvatting 117 Dankwoord 121 Curriculum Vitae 125 Abbreviations βTrCP beta-transducing repeat protein CIS cytokine-inducible SH2-domain containing protein DUB deubiquitinating enzyme EPOR Epo-receptor ER endoplasmic reticulum EV empty vector FERM four-point-one, ezrin, radixin, moesin GH Growth hormone GHR Growth hormone receptor GHBP Growth hormone binding protein GHRH growth hormone releasing hormone GHS growth hormone secretagogue HECT homologous to E6-AP C-terminus IGF insulin-like growth factor IκB Inhibitor of NFκB Jak Janus kinase NFκB Nuclear Factor κB MAPK mitogen activated protein kinase MVB multivesicular body PI3K phosphoinositide 3-kinase PKC protein kinase C PRL prolactin PTB phosphotyrosine binding PTP protein tyrosine phosphatase RING really interesting new gene SCF Skp-Cullin-F-box protein SH2 Src homology-2 SOCS suppressors of cytokine signalling STAT signal transducers and activators of transcription TACE tumor necrosis factor-receptor associated factor UbE ubiquitin-dependent endocytosis UBD ubiquitin-binding domain UBL ubiquitin-like protein UIM ubiquitin-interacting motif CHAPTER I General Introduction Joyce Putters Department of Cell Biology and Institute of Biomembranes University Medical Center Utrecht Chapter I Contents Chapter I Growth hormone............................................................................................9 Growth hormone family and structure GH secretion Clinical relevance of GH Growth hormone receptor............................................................................10 Cytokine superfamily GHR structure The ubiquitin proteasome system...............................................................14 Ubiquitin The function of protein ubiquitination The mechanism of protein ubiquitination SCF complexes History Structure and regulation of the SCF complex Mechanism and regulation of SCF complex activity SCFβTrCP Deubiquitination Ubiquitin-like proteins The ubiquitin system and disease GHR signalling...............................................................................................20 Turning the stimulus into a signal Jak family GHR signal transduction pathways The STAT pathway The MAPK pathway IRS, PI3K and NFκB Protein kinase C Regulation of GHR signalling Regulation by phosphatases and phosphorylation events Suppressors of cytokine signalling (SOCS) Other GHR signalling regulators Cytokine receptor trafficking.......................................................................25 The role of Jak2 in cytokine receptor trafficking The role of Jak2 in GHR trafficking The ubiquitin system and receptor trafficking The ubiquitin system and GHR trafficking Outline of this thesis.....................................................................................30 References.......................................................................................................31 8 Introduction Growth hormone Growth hormone family and structure Growth hormone (GH), also known as somatotropin or somatotrope hormone, belongs to a class of evolutionary related peptide hormones, e.g. prolactin (PRL) and placental lactogen (PL) (1). Although this class of peptide hormones does not share a high degree of sequential homology, the structures adopt very similar conformations. Furthermore, the structures of other cytokines such as erythropoietin (Epo), several interleukins and leptin are also analogous (2). Two main splice variants of GH have been identified, one of which contains 191 and the other 176 amino acids. 85% of circulating GH represents the 191 amino acid protein (3) and the structure of this GH variant has been crystallized more than 20 years ago (4). Generally, GH contains four α-helical domains connected by loops (5). In addition, two disulfide bonds were identified, of which the one connecting C53 to C165, is important for GH secretion, binding and biological activity (6). GH secretion The origin of GH in the circulation was found first to be the somatotrophic cells in the anterior pituitary gland (7), but to date several studies found that extra-pituitary tissues also produce GH, such as mammary (8), neuronal (9), retinal (10), lung (11), bone (12) and several types of immune cells (13,14). GH-levels in the circulation are regulated by several factors. Two of them are somatostatin, which inhibits GH-release, and GH- releasing hormone (GHRH), which stimulates GH-release. Furthermore, Growth Hormone Secretagogue Receptor (GHSR) induces GH release upon activation by the GH secretagogues (GHS), of which an example is ghrelin (15-17). Another important factor is pituitary transcription factor Pit-1, which regulates all GH synthesis and release, but is also involved in the differentiation and maintenance of GH-producing cells in the pituitary gland (18). Moreover, insulin-like growth factors (IGFs), which are induced by GH, inhibit GH-release, resulting in a negative feedback loop (19). The outcome of the many factors involved in the regulation of GH-release is a pulsatile GH-release pattern in the circulation (7). It is important to note that the pulsatile secretion pattern of GH is regulated by hypothalamic activity and is primarily aimed at the pituitary. The secretion regulation of other GH-producing cells, like in mammary tissue, is less well understood and not pulsatile. Therefore, female GH-secretion is not as strongly pulsatile compared to male GH-secretion (20,21), which results in sexual dimorphisms in several processes, including hepatic metabolism (22,23). GH has a half-life of about fifteen to twenty minutes and clearance occurs in the kidneys and by internalisation via the GH receptor (GHR). GH binding protein (GHBP), representing the extracellular domain of the GHR (24), circulating in the bloodstream is able to bind GH and prevents GH from being cleared from the circulation (25-27). In this way, GHBP can increase GH levels in the bloodstream, although this mechanism could also serve to let cells only respond to high GH levels that exceed the level of GHBP. Normally, about 50% of total GH in the bloodstream is in complex with GHBP (28). 9 Chapter I Clinical relevance of GH GH actions involve multiple organs and systems and are essential for postnatal longitudinal growth and development. Originally, it was postulated that all effects of GH were mediated by IGF1, which is known as ‘the somatomedin hypothesis’. His turned out not to be the case. Moreover, IGF1 expression is stimulated by many other factors than GH. IGF1’s most important role is in mediating the anabolic and linear growth promoting effect of GH (29). In addition to promoting postnatal longitudinal growth, GH is responsible for changes in protein, lipid and carbohydrate metabolism. Shortly, under starving conditions, GH changes consumption from carbohydrates and protein to lipids, which allows conservation of vital protein stores (30). GH hypersecretion results in acromegaly or gigantism, a condition associated with significant morbidity and mortality. Clinical features include coarse facial features, large, spade shaped hands and large feet. In contrast, GH hyposecretion results in dwarfism with clinical symptoms as reduced lean body mass, increased fat mass, impaired systolic function and impaired quality of life (29). Apart from GH secretion, defects in GHR, post-GHR receptor signalling or primary defects in IGF1 synthesis result in phenotypes resembling GH hyposecretion, varying from more or less severe. The first well-described clinical phenotype of severe growth failure was described by Laron and coworkers and therefore named the Laron syndrome (31). They initially searched for a defect in the GH protein and its secretion, but they found eventually that GH could not bind to the GHR due to deletions or mutations in the receptor (32,33). Growth hormone receptor Cytokine superfamily The GHR belongs to the class I cytokine