YGHIR-01073; No of Pages 5 Growth Hormone & IGF Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
Review article
The growth hormone receptor Michael J. Waters Institute for Molecular Bioscience, University of Queensland, St Lucia 4072, Australia
a r t i c l e
i n f o
Article history: Received 1 June 2015 Accepted 1 June 2015 Available online xxxx Keywords: JAK2 Mechanism Receptor structure STAT5 Src
a b s t r a c t Once thought to be present only in liver, muscle and adipose tissue, the GH receptor is now known to be ubiquitously distributed, in accord with the many pleiotropic actions of GH. These include the regulation of metabolism, postnatal growth, cognition, immune, cardiac and renal systems and gut function. GH exerts these actions primarily through alterations in gene expression, initiated by activation of its membrane receptor and the resultant activation of the associated JAK2 (Janus kinase 2) and Src family kinases. Receptor activation involves hormone initiated movements within a receptor homodimer, rather than simple receptor dimerization. We have shown that binding of the hormone realigns the orientation of the two receptors both by relative rotation and by closer apposition just above the cell membrane. This is a consequence of the asymmetric placement of the binding sites on the hormone. Binding results in a conversion of parallel receptor transmembrane domains into a rotated crossover orientation, which produces separation of the lower part of the transmembrane helices. Because the JAK2 is bound to the Box1 motif proximal to the inner membrane, receptor activation results in separation of the two associated JAK2s, and in particular the removal of the inhibitory pseudokinase domain from the kinase domain of the other JAK2 (and vice versa). This brings the two kinase domains into position for trans-activation and initiates tyrosine phosphorylation of the receptor cytoplasmic domain and other substrates such as STAT5, the key transcription factor mediating most genomic actions of GH. There are a limited number of genomic actions initiated by the Src kinase family member which also associates with the upper cytoplasmic domain of the receptor, including important immune regulatory actions to dampen exuberant innate immune activation of cells involved in transplant rejection. These findings offer insights for developing specific receptor antagonists which may be valuable in cancer therapy. Crown Copyright © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction While the postnatal growth enhancing actions of growth hormone (GH) are best known, GH also has a wide range of actions in the regulation of metabolism, (particularly in the liver and adipose tissue), as well as actions on the immune, reproductive and cardiovascular systems, and the brain [3]. For example, hepatic steatosis and gluconeogenesis are GH regulated, as are many cytochrome P450 enzymes involved in detoxification and drug metabolism. These have been best characterised in rodents, but the limited human evidence available supports human applicability. GH status is known to influence cognitive ability, and we have shown that GH is required for neural stem cell proliferation and formation of new neurons in response to exercise, while GH/IGF1 also influences synaptic connection formation [12]. In relation to immune function, GH influences many cellular immune elements, either directly or via IGF1 generation. For example, it is able to increase helper CD4 Tcell number in AIDS patients, and we have shown that GH induces expression of the mouse homolog of HLA-G, a key immune-tolerance
E-mail address: [email protected].
protein able to dampen the innate immune response [1]. Given the ubiquity of expression of the GH receptor (GHR) as demonstrated both by immunohistochemical means and by transcript analysis, it was evident that GH would have actions in every tissue of the body, and this has subsequently been verified by many studies [3]. It is important to understand that GH acts through mediators for many of its actions. Thus, as well as induction of IGF1, GH is able to upregulate the EGF, oestrogen and androgen receptors, and the angiotensin II receptor, and we showed that GH upregulates the expression of BMP2/4 and the BMP 1A receptor [3,10,18]. The actions of GH are a result of activation of its receptor, and loss of function of this receptor has, as one would surmise from the above, many consequences. Because GH is a modulator, such a loss is not lethal, but results in a sub optimal health with short stature, decreased bone mineral density and concentration, decreased muscle strength, thin skin and hair, delayed puberty and increased adiposity, and hepatic steatosis, along with impaired cognitive ability in several fields [3]. Interestingly, these patients are highly resistant to cancer, and in the Ecuadorian cohort of 100, no cancer deaths were observed [8]. Conversely, prolonged activation of the receptor as a result of a variant sequence in its cytoplasmic domain which impairs the binding of the
http://dx.doi.org/10.1016/j.ghir.2015.06.001 1096-6374/Crown Copyright © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
2
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx
SOCS2 negative regulator of GH action, is associated with an increased risk from lung cancer [7]. 2. Structure of the GH receptor Given the importance of its physiologic actions, what do we know of the GH receptor itself? This receptor was the first class 1 cytokine receptor to be cloned, and has been an exemplar for the 30 receptors in this class, which include the receptors for erythropoietin, prolactin, leptin, thrombopoietin, LIF, CTNF, oncostatin-M, carditropin-1 and most of the interleukins, together with many of the haematopoietic colonystimulating factors. These form homodimers or heterodimers with accessory proteins such as gp 130, common beta and common gamma chains (Fig. 1 and ref. [16]). They are all single membrane pass receptors possessing a characteristic domain structure in their extracellular domains referred to as a cytokine receptor homology domain, which consists of two fibronectin III like modules, each with a seven stranded beta sandwich structure which is the focus of ligand binding. A conserved WSxWS motif is present in the lower FNIII module (YGeFS for the GHR), necessary for expression and stability of these receptors. Many of these receptors release their extracellular domain into the circulation, and in the case of the GHR this produces the circulating GH binding protein, which regulates GH availability [2]. The key common element for this class of receptor is the conserved proline rich Box 1 motif, which is necessary for binding of their cognate tyrosine (Janus) kinase, the main target of the receptor activation process. This is located close to the cell membrane, and N-terminal to the less conserved Box 2 sequence consisting of aromatic and acidic residues. The Janus kinases phosphorylate not only certain tyrosine residues in the receptor cytoplasmic domain, but also other protein substrates directly. In the case of the GHR, key tyrosine residues in the cytoplasmic domain serve as docking sites for SH2-domain containing proteins, notably STAT5a and STAT5b, which mediate a considerable part of GH action at the genome. Docking of these transcription factors facilitates their phosphorylation by the JAK2 at tyrosine 699, resulting in STAT5 dimer formation and translocation to the nucleus where the dimer regulates target gene
expression [11]. Recently we elucidated the structure of the cytoplasmic domain of both the GHR and the prolactin receptor, and found them to be intrinsically disordered, with some transient alpha-helical structure [9]. Intrinsically disordered structure was also observed for the shared gp130 subunit of other class 1 receptors and would confer maximal flexibility on the cytoplasmic domain, allowing it to bind multiple proteins as well as access to the membrane proximal JAK2 for phosphorylation of its target tyrosines. 3. Activation of the GH receptor While it is generally believed that class 1 cytokine receptors are activated by ligand-dependent dimerization/oligomerization, we and others have shown that the GH, EPO and prolactin receptors exist as dimers at the cell surface before binding of the cognate ligand (reviewed in [16]). Using receptor truncations with FRET and BRET reporters at the N- or C-terminus, we showed that the major element responsible for dimerization of the unliganded receptor was the helical transmembrane domains. This has necessitated a reappraisal of the receptor activation mechanism, focusing on ligand-induced conformational change or subunit realignment (see [17] for an account of the history). Initially we sought ligand dependent changes in the extracellular domain by comparing the Genentech crystal structure of the GH(receptor)2 structure with our unliganded receptor crystal structure [5]. However, this revealed no substantial difference in disposition of the peptide chain between receptor 1 in the ternary complex and the unliganded receptor. Hence the remaining option: realignment of the receptors as a result of binding the hormone which possesses two binding sites, placed asymmetrically. Our previous mutational studies had shown that the receptor–receptor interaction surface in the extracellular domain (site 3, involving the lower Fibronectin III domain) must lock together for signalling to occur [6]. Evidently the asymmetric placement of the receptor binding sites on the hormone aligns these site 3 surfaces, so they can lock together. Evidence for such a realignment was published by Poger and Mark [13], wherein in silico removal of the hormone from the GH(receptor)2 complex in GROMOS resulted in relative
Fig. 1. Domain structures of class 1 cytokine receptors showing disulphide bonded domain 1 (yellow) and domain 2 (red), with Box 1 and 2 sequences wand the conserved WSxWS motif. Common subunits shared by heteromeric receptors also shown.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx
rotation of the two receptor subunits by around 45°, representing the putative unliganded state of the dimer. We explored the role of receptor realignment by replacing the extracellular domain of the receptor with a jun zipper, to clamp the receptors in specific alignments [4]. We used two parallel sets of constructs, one with the complete cytoplasmic domain to observe signalling to STAT5, and a second with a FRET reporter place below the Box 1 motif. In this way we could correlate movements of the Box 1 motif with receptor activation. We found that the closer the zippers were to the cell surface, the stronger was the STAT5 activation signal. There was also an effect of rotation, with a maximum activation at a particular rotational position conferred by insertion of alanine residues at the membrane surface. We then introduced a charge reversal into the highly conserved EED sequence just above the membrane, and found that combining EED with KKR markedly increased the STAT5 activation (this was also effective with the full length native receptor). They key point here is that with each of these strategies there was an INVERSE relationship between activation and the FRET signal ie the more active constructs resulted in INCREASED separation of the Box 1 sequences. We tested this surprising conclusion in the full length receptor with hGH itself, then with the charge reversal approach, then using the GH antagonist hGHG120R, and finally the Laron dwarf mutation D170H in Site 3, which does not influence hormone binding, but does block signalling. In each case we ran a parallel set of constructs with the C-terminal FRET reporters to monitor movement of Box 1. In each case the inverse relation was observed between activity and FRET signal, with the G120R blocking the ability of hGH to decrease the FRET signal, while the D170H receptor mutation did not show the decrease in FRET signal seen on addition of hGH. To understand the movements of the transmembrane domains (TMDs) necessary to separate the Box 1 sequences upon receptor activation, we took two approaches [4]. In the first, we converted every TMD residue, as well as nearby juxtamembrane residues, individually to cysteine. We then crosslinked these using a short crosslinker and ran non-reducing SDS gels to separate monomer and dimer for each residue substitution, thus providing proximity estimates for individual residues. This provided us with the relationship between the two TM helices in the dimer in the unliganded (basal) state. We had hoped to see a difference on addition of ligand (hormone), but the helix movement on activation was insufficient to show in the crosslinking, except at the cell surface. These studies did however, provide us with the
3
basal state for the molecular dynamics modelling we the then undertook. For these studies, we placed the TMDs in a lipid bilayer (DPPC) and moved them slowly together, observing their orientation and resulting free energies. The helices interacted strongly, driven by entropic and lipid interactions, initially in a parallel arrangement as seen with the crosslinking studies for the unliganded receptor. Further approach, as would be a consequence of locking of Site 3 through hormone binding, converted the parallel form to a left handed crossover form, with separation of the C-termini of the helices, as observed in the FRET studies (Fig. 2). This movement to a crossover arrangement required a rotation of helices resulting in close contact of two conserved glycine residues in the centre of the TMD, with removal of two phenylalanine residues from the interaction surface of the parallel dimer. It was gratifying that when we docked the active helix crossover form with the crystal structure of the 1:2 complex, the fit was facile, and resulted in close proximity of Cys 259 in the upper juxtamembrane segment, in accordance with the formation of a spontaneous disulphide bond on hormone binding. 4. Activation of JAK2 Having established the helix movements involved in receptor activation, the next task was to understand how a separation of Box 1 motifs could activate the associated JAK2s, which work in tandem. A key point here is that these tyrosine kinases possess, unusually, a pseudokinase inhibitory domain. This must be removed from the catalytic kinase domain for signalling to occur. We hypothesised that if the pseudokinase domain of one JAK2 was inhibiting the kinase domain of the other JAK2, then a separation of Box 1 motifs could slide the pseudokinase domain away from the other kinase domain, and bring the two kinase domains into proximity for trans-activation to occur ([4], see Fig. 3). Evidence for this model was first obtained by placing FRET reporters at the positions of the kinase domain and at the pseudokinase domain. We used the charge reversal mutant to avoid the problem of cryptic receptors in membrane vesicles without access to the hormone. It was observed that when the receptor became activated the pseudokinase domains became more separated (lower FRET signal), while the kinase domain FRET reporters gave a stronger signal, as predicted. Second, we swapped the kinase and pseudokinase domains, reasoning that if the domain swap was co-transfected with the native JAK2, this would place the kinase domains together, conferring constitutive activity.
Fig. 2. Transmembrane helix rearrangement upon activation of the receptor, derived from molecular dynamics simulations in a lipid bilayer. The basal state (parallel helices) converts to the active form by closer approach of the N-termini, leading to a rotation and left hand crossover of the helices with separation of the C-termini.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
4
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx
Fig. 3. Schematic of activation process for JAK2, driven by separation of the receptor C-termini. This removes the inhibitory pseudokinase of one JAK2 from the kinase domain of the other JAK2 (and vice versa), relieving inhibition and bringing the kinase domains together for trans-activation.
This was indeed observed in 3 separate experiments. Third, we showed association of pseudokinase–kinase pairs by Alpha screen and single molecule fluorescence coincidence measurement, while the kinase domains alone did not associate. Finally, we docked the crystal structures of the kinase and pseudokinase domains of JAK2 and found a minimum energy configuration that allowed for orientations satisfying our Box1 separation hypothesis. This configuration could also explain the oncogenicity of several known JAK2 mutations, including the V617F mutant evident in polycythemia vera. Together, these findings are illustrated in a flash animation showing the activation process: http://web-services.imb.uq.edu.au/waters/hgh. html. 5. In vivo analysis of signalling by the GHR and Src family kinases Some years ago, when the nature of the signal for postnatal growth was unknown, we sought to identify it with targeted knockin mutations in the cytoplasmic domain of the receptor, using mice. From a series of truncations we correlated postnatal growth with ability to activate STAT5, and identified GH-regulated transcripts associated with signalling from the different parts of the receptor cytoplasmic domain [14]. These mice have also allowed us to identify the elements involved in the ability of GH to promote browning of white adipose tissue, as part of its ability to decrease adiposity. They have also been useful in identifying elements responsible for the insulin-antagonistic actions of GH. In addition to the receptor truncations referred to above, we made a knockin mutant with Box 1 disabled by alanine substitution of the 4 prolines [15]. The dwarf mutant was unable to activate JAK2 or STAT5 in response to GH administration, but was able to activate Src signalling to ERK. This was in agreement with in vitro studies we had performed showing that a Src family kinase (SFK) associates with the GHR, and can signal independently of JAK2 with a different orientation of the TMD, in this case leading to activation of ERK and Jun [15]. The physiological consequences of this were unclear until we performed microarray analysis of the transcripts in the livers of these mice, and compared the profile with that of the GHRKO [1]. We identified transcripts specific to the Src-ERK-Jun pathway which included the mouse homolog of a potent immunotolerance protein known as HLA-G. Serum levels of this protein correlate with success of renal, cardiac, lung and liver transplants in humans, and it is used to protect the foetus from attack by the maternal immune system. We were able to show that this protein is
required for liver regeneration in the partial hepatectomy model with C567Bl/6 mice, and that this gene is induced by the Src-ERK-Jun pathway in response to GH. Its function is to dampen the innate immune system which is triggered by the sterile inflammatory response produced by the surgical insult of partial hepatectomy. Replacement of this gene both by adenoviral delivery and by direct infusion of the protein prevented these mice from dying within 48 h of the operation and suppressed markers of immune attack. 6. Conclusion The GH receptor uses two tyrosine kinases for its signalling, and they are activated by different conformational changes within the constitutive dimer. We have elucidated the activation mechanism for JAK2, and are currently defining the mechanism for the SFK component of signalling. It is anticipated this information will be of use for drug design purposes, particularly for the creation of a GH antagonist which may be useful in cancer therapy. I have no conflicts of interest associated with this review manuscript. Acknowledgements Supported by NHMRC (Australia) grants 511120, 1002893, 1025088, 1025082 to MJW. References [1] J.L. Barclay, L.M. Kerr, L. Arthur, J.E. Rowland, C.N. Nelson, M. Ishikawa, E.M. d'Aniello, M. White, P.G. Noakes, M.J. Waters, In vivo targeting of the growth hormone receptor (GHR) Box1 sequence demonstrates that the GHR does not signal exclusively through JAK2, Mol. Endocrinol. 24 (2010) 204–217. [2] R. Barnard, M.J. Waters, The serum GH binding protein: pregnant with possibilities, J. Endocrinol. 153 (1997) 1–14. [3] A.J. Brooks, M.J. Waters, The growth hormone receptor: mechanism of activation and clinical implications, Nat. Rev. Endocrinol. 6 (2010) 515–525. [4] A.J. Brooks, W. Dai, M.L. O'Mara, D. Abankwa, Y. Chhabra, R.A. Pelekanos, O. Gardon, K.A. Tunny, K.M. Blucher, C.J. Morton, M.W. Parker, E. Sierecki, Y. Gambin, G.A. Gomez, K. Alexandrov, I.A. Wilson, M. Doxastakis, A.E. Mark, M.J. Waters, Science 344 (6185) (2014) 1249783. [5] R.J. Brown, J.J. Adams, R.A. Pelekanos, Y. Wan, W.J. McKinstry, K. Palethorpe, R.M. Seeber, T.A. Monks, K.A. Eidne, M.W. Parker, M.J. Waters, Model for growth hormone receptor activation based on subunit rotation within a receptor dimer, Nat. Struct. Mol. Biol. 12 (2005) 814–821. [6] C.-M. Chen, R. Brinkworth, M.J. Waters, The role of receptor dimerization domain residues in GH signalling, J. Biol. Chem. 272 (1997) (1997) 5133–5140.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx [7] Y. Chhabra, A.J. Brooks, M.J. Waters, The first cancer-associated variant of the growth hormone receptor, Endocrine Society Proceedings, Chicago2014. (abstract 13698). [8] J. Guevara-Aguirre, P. Balasubramanian, M. Guevara-Aguirre, M. Wei, F. Madia, C.W. Cheng, D. Hwang, A. Martin-Montalvo, J. Saavedra, S. Ingles, R. de Cabo, P. Cohen, V.D. Longo, Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans, Sci. Transl. Med. 3 (2011) (70ra13). [9] G.W. Haxholm, L.F. Nikolajsen, J.G. Olsen, J. Fredsted, F.H. Larsen, V. Goffin, S.F. Pedersen, A.J. Brooks, M.J. Waters, B.B. Kragelund, Biochem. J. (Apr 7 2015), http:// dx.doi.org/10.1042/BJ20141243 (epub). [10] H. Li, P.M. Bartold, C.Z. Zhang, R.W. Clarkson, W.G. Young, M.J. Waters, GH and IGF-1 induce bone morphogenetic proteins 2 and 4: a mediator role in bone and tooth formation? Endocrinology 139 (1998) 3855–3862. [11] A.M. Lichanska, M.J. Waters, How growth hormone controls growth, obesity and sexual dimorphism, Trends Genet. 24 (2008) 41–47. [12] F. Nyberg, M. Hallberg, Growth hormone and cognitive function, Nat. Rev. Endocrinol. 9 (2013) 357–365.
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[13] D. Poger, A.E. Mark, Turning the growth hormone receptor on: evidence that hormone binding induces subunit rotation, Proteins 78 (2010) 1163–1174. [14] J.E. Rowland, A.M. Lichanska, L.M. Kerr, M. White, E. D'Aniello, S.L. Maher, R.J. Brown, R. Teasdale, P.G. Noakes, M.J. Waters, In vivo analysis of growth hormone receptor signalling domains and their associated transcripts, Mol. Cell. Biol. 25 (2006) 66–77. [15] S.W. Rowlinson, H. Yoshizato, J.L. Barclay, A.J. Brooks, S.N. Behncken, L.M. Kerr, K. Millard, K. Palethorpe, K. Nielsen, J. Clyde-Smith, J.F. Hancock, M.J. Waters, An agonist-induced conformational change in the growth hormone receptor determines the choice of signalling pathway, Nat. Cell Biol. 10 (2008) 740–747. [16] M.J. Waters, C.A. Shang, S.N. Behncken, S.-P. Tam, H. Li, B. Shen, P.E. Lobie, Growth hormone as a cytokine, Clin. Exp. Pharmacol. Physiol. 26 (1999) 760–764. [17] M.J. Waters, A.J. Brooks, JAK2 activation by growth hormone and other cytokines, Biochem. J. 466 (2015) 1–11. [18] M.J. Waters, A.J. Brooks, Y. Chhabra, A new mechanism for growth hormone receptor activation of JAK2, and implications for related cytokine receptors, JAKSTAT 3 (2014) e29569.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
Contents lists available at ScienceDirect
Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
Review article
The growth hormone receptor Michael J. Waters Institute for Molecular Bioscience, University of Queensland, St Lucia 4072, Australia
a r t i c l e
i n f o
Article history: Received 1 June 2015 Accepted 1 June 2015 Available online xxxx Keywords: JAK2 Mechanism Receptor structure STAT5 Src
a b s t r a c t Once thought to be present only in liver, muscle and adipose tissue, the GH receptor is now known to be ubiquitously distributed, in accord with the many pleiotropic actions of GH. These include the regulation of metabolism, postnatal growth, cognition, immune, cardiac and renal systems and gut function. GH exerts these actions primarily through alterations in gene expression, initiated by activation of its membrane receptor and the resultant activation of the associated JAK2 (Janus kinase 2) and Src family kinases. Receptor activation involves hormone initiated movements within a receptor homodimer, rather than simple receptor dimerization. We have shown that binding of the hormone realigns the orientation of the two receptors both by relative rotation and by closer apposition just above the cell membrane. This is a consequence of the asymmetric placement of the binding sites on the hormone. Binding results in a conversion of parallel receptor transmembrane domains into a rotated crossover orientation, which produces separation of the lower part of the transmembrane helices. Because the JAK2 is bound to the Box1 motif proximal to the inner membrane, receptor activation results in separation of the two associated JAK2s, and in particular the removal of the inhibitory pseudokinase domain from the kinase domain of the other JAK2 (and vice versa). This brings the two kinase domains into position for trans-activation and initiates tyrosine phosphorylation of the receptor cytoplasmic domain and other substrates such as STAT5, the key transcription factor mediating most genomic actions of GH. There are a limited number of genomic actions initiated by the Src kinase family member which also associates with the upper cytoplasmic domain of the receptor, including important immune regulatory actions to dampen exuberant innate immune activation of cells involved in transplant rejection. These findings offer insights for developing specific receptor antagonists which may be valuable in cancer therapy. Crown Copyright © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction While the postnatal growth enhancing actions of growth hormone (GH) are best known, GH also has a wide range of actions in the regulation of metabolism, (particularly in the liver and adipose tissue), as well as actions on the immune, reproductive and cardiovascular systems, and the brain [3]. For example, hepatic steatosis and gluconeogenesis are GH regulated, as are many cytochrome P450 enzymes involved in detoxification and drug metabolism. These have been best characterised in rodents, but the limited human evidence available supports human applicability. GH status is known to influence cognitive ability, and we have shown that GH is required for neural stem cell proliferation and formation of new neurons in response to exercise, while GH/IGF1 also influences synaptic connection formation [12]. In relation to immune function, GH influences many cellular immune elements, either directly or via IGF1 generation. For example, it is able to increase helper CD4 Tcell number in AIDS patients, and we have shown that GH induces expression of the mouse homolog of HLA-G, a key immune-tolerance
E-mail address: [email protected].
protein able to dampen the innate immune response [1]. Given the ubiquity of expression of the GH receptor (GHR) as demonstrated both by immunohistochemical means and by transcript analysis, it was evident that GH would have actions in every tissue of the body, and this has subsequently been verified by many studies [3]. It is important to understand that GH acts through mediators for many of its actions. Thus, as well as induction of IGF1, GH is able to upregulate the EGF, oestrogen and androgen receptors, and the angiotensin II receptor, and we showed that GH upregulates the expression of BMP2/4 and the BMP 1A receptor [3,10,18]. The actions of GH are a result of activation of its receptor, and loss of function of this receptor has, as one would surmise from the above, many consequences. Because GH is a modulator, such a loss is not lethal, but results in a sub optimal health with short stature, decreased bone mineral density and concentration, decreased muscle strength, thin skin and hair, delayed puberty and increased adiposity, and hepatic steatosis, along with impaired cognitive ability in several fields [3]. Interestingly, these patients are highly resistant to cancer, and in the Ecuadorian cohort of 100, no cancer deaths were observed [8]. Conversely, prolonged activation of the receptor as a result of a variant sequence in its cytoplasmic domain which impairs the binding of the
http://dx.doi.org/10.1016/j.ghir.2015.06.001 1096-6374/Crown Copyright © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
2
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx
SOCS2 negative regulator of GH action, is associated with an increased risk from lung cancer [7]. 2. Structure of the GH receptor Given the importance of its physiologic actions, what do we know of the GH receptor itself? This receptor was the first class 1 cytokine receptor to be cloned, and has been an exemplar for the 30 receptors in this class, which include the receptors for erythropoietin, prolactin, leptin, thrombopoietin, LIF, CTNF, oncostatin-M, carditropin-1 and most of the interleukins, together with many of the haematopoietic colonystimulating factors. These form homodimers or heterodimers with accessory proteins such as gp 130, common beta and common gamma chains (Fig. 1 and ref. [16]). They are all single membrane pass receptors possessing a characteristic domain structure in their extracellular domains referred to as a cytokine receptor homology domain, which consists of two fibronectin III like modules, each with a seven stranded beta sandwich structure which is the focus of ligand binding. A conserved WSxWS motif is present in the lower FNIII module (YGeFS for the GHR), necessary for expression and stability of these receptors. Many of these receptors release their extracellular domain into the circulation, and in the case of the GHR this produces the circulating GH binding protein, which regulates GH availability [2]. The key common element for this class of receptor is the conserved proline rich Box 1 motif, which is necessary for binding of their cognate tyrosine (Janus) kinase, the main target of the receptor activation process. This is located close to the cell membrane, and N-terminal to the less conserved Box 2 sequence consisting of aromatic and acidic residues. The Janus kinases phosphorylate not only certain tyrosine residues in the receptor cytoplasmic domain, but also other protein substrates directly. In the case of the GHR, key tyrosine residues in the cytoplasmic domain serve as docking sites for SH2-domain containing proteins, notably STAT5a and STAT5b, which mediate a considerable part of GH action at the genome. Docking of these transcription factors facilitates their phosphorylation by the JAK2 at tyrosine 699, resulting in STAT5 dimer formation and translocation to the nucleus where the dimer regulates target gene
expression [11]. Recently we elucidated the structure of the cytoplasmic domain of both the GHR and the prolactin receptor, and found them to be intrinsically disordered, with some transient alpha-helical structure [9]. Intrinsically disordered structure was also observed for the shared gp130 subunit of other class 1 receptors and would confer maximal flexibility on the cytoplasmic domain, allowing it to bind multiple proteins as well as access to the membrane proximal JAK2 for phosphorylation of its target tyrosines. 3. Activation of the GH receptor While it is generally believed that class 1 cytokine receptors are activated by ligand-dependent dimerization/oligomerization, we and others have shown that the GH, EPO and prolactin receptors exist as dimers at the cell surface before binding of the cognate ligand (reviewed in [16]). Using receptor truncations with FRET and BRET reporters at the N- or C-terminus, we showed that the major element responsible for dimerization of the unliganded receptor was the helical transmembrane domains. This has necessitated a reappraisal of the receptor activation mechanism, focusing on ligand-induced conformational change or subunit realignment (see [17] for an account of the history). Initially we sought ligand dependent changes in the extracellular domain by comparing the Genentech crystal structure of the GH(receptor)2 structure with our unliganded receptor crystal structure [5]. However, this revealed no substantial difference in disposition of the peptide chain between receptor 1 in the ternary complex and the unliganded receptor. Hence the remaining option: realignment of the receptors as a result of binding the hormone which possesses two binding sites, placed asymmetrically. Our previous mutational studies had shown that the receptor–receptor interaction surface in the extracellular domain (site 3, involving the lower Fibronectin III domain) must lock together for signalling to occur [6]. Evidently the asymmetric placement of the receptor binding sites on the hormone aligns these site 3 surfaces, so they can lock together. Evidence for such a realignment was published by Poger and Mark [13], wherein in silico removal of the hormone from the GH(receptor)2 complex in GROMOS resulted in relative
Fig. 1. Domain structures of class 1 cytokine receptors showing disulphide bonded domain 1 (yellow) and domain 2 (red), with Box 1 and 2 sequences wand the conserved WSxWS motif. Common subunits shared by heteromeric receptors also shown.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx
rotation of the two receptor subunits by around 45°, representing the putative unliganded state of the dimer. We explored the role of receptor realignment by replacing the extracellular domain of the receptor with a jun zipper, to clamp the receptors in specific alignments [4]. We used two parallel sets of constructs, one with the complete cytoplasmic domain to observe signalling to STAT5, and a second with a FRET reporter place below the Box 1 motif. In this way we could correlate movements of the Box 1 motif with receptor activation. We found that the closer the zippers were to the cell surface, the stronger was the STAT5 activation signal. There was also an effect of rotation, with a maximum activation at a particular rotational position conferred by insertion of alanine residues at the membrane surface. We then introduced a charge reversal into the highly conserved EED sequence just above the membrane, and found that combining EED with KKR markedly increased the STAT5 activation (this was also effective with the full length native receptor). They key point here is that with each of these strategies there was an INVERSE relationship between activation and the FRET signal ie the more active constructs resulted in INCREASED separation of the Box 1 sequences. We tested this surprising conclusion in the full length receptor with hGH itself, then with the charge reversal approach, then using the GH antagonist hGHG120R, and finally the Laron dwarf mutation D170H in Site 3, which does not influence hormone binding, but does block signalling. In each case we ran a parallel set of constructs with the C-terminal FRET reporters to monitor movement of Box 1. In each case the inverse relation was observed between activity and FRET signal, with the G120R blocking the ability of hGH to decrease the FRET signal, while the D170H receptor mutation did not show the decrease in FRET signal seen on addition of hGH. To understand the movements of the transmembrane domains (TMDs) necessary to separate the Box 1 sequences upon receptor activation, we took two approaches [4]. In the first, we converted every TMD residue, as well as nearby juxtamembrane residues, individually to cysteine. We then crosslinked these using a short crosslinker and ran non-reducing SDS gels to separate monomer and dimer for each residue substitution, thus providing proximity estimates for individual residues. This provided us with the relationship between the two TM helices in the dimer in the unliganded (basal) state. We had hoped to see a difference on addition of ligand (hormone), but the helix movement on activation was insufficient to show in the crosslinking, except at the cell surface. These studies did however, provide us with the
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basal state for the molecular dynamics modelling we the then undertook. For these studies, we placed the TMDs in a lipid bilayer (DPPC) and moved them slowly together, observing their orientation and resulting free energies. The helices interacted strongly, driven by entropic and lipid interactions, initially in a parallel arrangement as seen with the crosslinking studies for the unliganded receptor. Further approach, as would be a consequence of locking of Site 3 through hormone binding, converted the parallel form to a left handed crossover form, with separation of the C-termini of the helices, as observed in the FRET studies (Fig. 2). This movement to a crossover arrangement required a rotation of helices resulting in close contact of two conserved glycine residues in the centre of the TMD, with removal of two phenylalanine residues from the interaction surface of the parallel dimer. It was gratifying that when we docked the active helix crossover form with the crystal structure of the 1:2 complex, the fit was facile, and resulted in close proximity of Cys 259 in the upper juxtamembrane segment, in accordance with the formation of a spontaneous disulphide bond on hormone binding. 4. Activation of JAK2 Having established the helix movements involved in receptor activation, the next task was to understand how a separation of Box 1 motifs could activate the associated JAK2s, which work in tandem. A key point here is that these tyrosine kinases possess, unusually, a pseudokinase inhibitory domain. This must be removed from the catalytic kinase domain for signalling to occur. We hypothesised that if the pseudokinase domain of one JAK2 was inhibiting the kinase domain of the other JAK2, then a separation of Box 1 motifs could slide the pseudokinase domain away from the other kinase domain, and bring the two kinase domains into proximity for trans-activation to occur ([4], see Fig. 3). Evidence for this model was first obtained by placing FRET reporters at the positions of the kinase domain and at the pseudokinase domain. We used the charge reversal mutant to avoid the problem of cryptic receptors in membrane vesicles without access to the hormone. It was observed that when the receptor became activated the pseudokinase domains became more separated (lower FRET signal), while the kinase domain FRET reporters gave a stronger signal, as predicted. Second, we swapped the kinase and pseudokinase domains, reasoning that if the domain swap was co-transfected with the native JAK2, this would place the kinase domains together, conferring constitutive activity.
Fig. 2. Transmembrane helix rearrangement upon activation of the receptor, derived from molecular dynamics simulations in a lipid bilayer. The basal state (parallel helices) converts to the active form by closer approach of the N-termini, leading to a rotation and left hand crossover of the helices with separation of the C-termini.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
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M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx
Fig. 3. Schematic of activation process for JAK2, driven by separation of the receptor C-termini. This removes the inhibitory pseudokinase of one JAK2 from the kinase domain of the other JAK2 (and vice versa), relieving inhibition and bringing the kinase domains together for trans-activation.
This was indeed observed in 3 separate experiments. Third, we showed association of pseudokinase–kinase pairs by Alpha screen and single molecule fluorescence coincidence measurement, while the kinase domains alone did not associate. Finally, we docked the crystal structures of the kinase and pseudokinase domains of JAK2 and found a minimum energy configuration that allowed for orientations satisfying our Box1 separation hypothesis. This configuration could also explain the oncogenicity of several known JAK2 mutations, including the V617F mutant evident in polycythemia vera. Together, these findings are illustrated in a flash animation showing the activation process: http://web-services.imb.uq.edu.au/waters/hgh. html. 5. In vivo analysis of signalling by the GHR and Src family kinases Some years ago, when the nature of the signal for postnatal growth was unknown, we sought to identify it with targeted knockin mutations in the cytoplasmic domain of the receptor, using mice. From a series of truncations we correlated postnatal growth with ability to activate STAT5, and identified GH-regulated transcripts associated with signalling from the different parts of the receptor cytoplasmic domain [14]. These mice have also allowed us to identify the elements involved in the ability of GH to promote browning of white adipose tissue, as part of its ability to decrease adiposity. They have also been useful in identifying elements responsible for the insulin-antagonistic actions of GH. In addition to the receptor truncations referred to above, we made a knockin mutant with Box 1 disabled by alanine substitution of the 4 prolines [15]. The dwarf mutant was unable to activate JAK2 or STAT5 in response to GH administration, but was able to activate Src signalling to ERK. This was in agreement with in vitro studies we had performed showing that a Src family kinase (SFK) associates with the GHR, and can signal independently of JAK2 with a different orientation of the TMD, in this case leading to activation of ERK and Jun [15]. The physiological consequences of this were unclear until we performed microarray analysis of the transcripts in the livers of these mice, and compared the profile with that of the GHRKO [1]. We identified transcripts specific to the Src-ERK-Jun pathway which included the mouse homolog of a potent immunotolerance protein known as HLA-G. Serum levels of this protein correlate with success of renal, cardiac, lung and liver transplants in humans, and it is used to protect the foetus from attack by the maternal immune system. We were able to show that this protein is
required for liver regeneration in the partial hepatectomy model with C567Bl/6 mice, and that this gene is induced by the Src-ERK-Jun pathway in response to GH. Its function is to dampen the innate immune system which is triggered by the sterile inflammatory response produced by the surgical insult of partial hepatectomy. Replacement of this gene both by adenoviral delivery and by direct infusion of the protein prevented these mice from dying within 48 h of the operation and suppressed markers of immune attack. 6. Conclusion The GH receptor uses two tyrosine kinases for its signalling, and they are activated by different conformational changes within the constitutive dimer. We have elucidated the activation mechanism for JAK2, and are currently defining the mechanism for the SFK component of signalling. It is anticipated this information will be of use for drug design purposes, particularly for the creation of a GH antagonist which may be useful in cancer therapy. I have no conflicts of interest associated with this review manuscript. Acknowledgements Supported by NHMRC (Australia) grants 511120, 1002893, 1025088, 1025082 to MJW. References [1] J.L. Barclay, L.M. Kerr, L. Arthur, J.E. Rowland, C.N. Nelson, M. Ishikawa, E.M. d'Aniello, M. White, P.G. Noakes, M.J. Waters, In vivo targeting of the growth hormone receptor (GHR) Box1 sequence demonstrates that the GHR does not signal exclusively through JAK2, Mol. Endocrinol. 24 (2010) 204–217. [2] R. Barnard, M.J. Waters, The serum GH binding protein: pregnant with possibilities, J. Endocrinol. 153 (1997) 1–14. [3] A.J. Brooks, M.J. Waters, The growth hormone receptor: mechanism of activation and clinical implications, Nat. Rev. Endocrinol. 6 (2010) 515–525. [4] A.J. Brooks, W. Dai, M.L. O'Mara, D. Abankwa, Y. Chhabra, R.A. Pelekanos, O. Gardon, K.A. Tunny, K.M. Blucher, C.J. Morton, M.W. Parker, E. Sierecki, Y. Gambin, G.A. Gomez, K. Alexandrov, I.A. Wilson, M. Doxastakis, A.E. Mark, M.J. Waters, Science 344 (6185) (2014) 1249783. [5] R.J. Brown, J.J. Adams, R.A. Pelekanos, Y. Wan, W.J. McKinstry, K. Palethorpe, R.M. Seeber, T.A. Monks, K.A. Eidne, M.W. Parker, M.J. Waters, Model for growth hormone receptor activation based on subunit rotation within a receptor dimer, Nat. Struct. Mol. Biol. 12 (2005) 814–821. [6] C.-M. Chen, R. Brinkworth, M.J. Waters, The role of receptor dimerization domain residues in GH signalling, J. Biol. Chem. 272 (1997) (1997) 5133–5140.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
M.J. Waters / Growth Hormone & IGF Research xxx (2015) xxx–xxx [7] Y. Chhabra, A.J. Brooks, M.J. Waters, The first cancer-associated variant of the growth hormone receptor, Endocrine Society Proceedings, Chicago2014. (abstract 13698). [8] J. Guevara-Aguirre, P. Balasubramanian, M. Guevara-Aguirre, M. Wei, F. Madia, C.W. Cheng, D. Hwang, A. Martin-Montalvo, J. Saavedra, S. Ingles, R. de Cabo, P. Cohen, V.D. Longo, Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans, Sci. Transl. Med. 3 (2011) (70ra13). [9] G.W. Haxholm, L.F. Nikolajsen, J.G. Olsen, J. Fredsted, F.H. Larsen, V. Goffin, S.F. Pedersen, A.J. Brooks, M.J. Waters, B.B. Kragelund, Biochem. J. (Apr 7 2015), http:// dx.doi.org/10.1042/BJ20141243 (epub). [10] H. Li, P.M. Bartold, C.Z. Zhang, R.W. Clarkson, W.G. Young, M.J. Waters, GH and IGF-1 induce bone morphogenetic proteins 2 and 4: a mediator role in bone and tooth formation? Endocrinology 139 (1998) 3855–3862. [11] A.M. Lichanska, M.J. Waters, How growth hormone controls growth, obesity and sexual dimorphism, Trends Genet. 24 (2008) 41–47. [12] F. Nyberg, M. Hallberg, Growth hormone and cognitive function, Nat. Rev. Endocrinol. 9 (2013) 357–365.
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[13] D. Poger, A.E. Mark, Turning the growth hormone receptor on: evidence that hormone binding induces subunit rotation, Proteins 78 (2010) 1163–1174. [14] J.E. Rowland, A.M. Lichanska, L.M. Kerr, M. White, E. D'Aniello, S.L. Maher, R.J. Brown, R. Teasdale, P.G. Noakes, M.J. Waters, In vivo analysis of growth hormone receptor signalling domains and their associated transcripts, Mol. Cell. Biol. 25 (2006) 66–77. [15] S.W. Rowlinson, H. Yoshizato, J.L. Barclay, A.J. Brooks, S.N. Behncken, L.M. Kerr, K. Millard, K. Palethorpe, K. Nielsen, J. Clyde-Smith, J.F. Hancock, M.J. Waters, An agonist-induced conformational change in the growth hormone receptor determines the choice of signalling pathway, Nat. Cell Biol. 10 (2008) 740–747. [16] M.J. Waters, C.A. Shang, S.N. Behncken, S.-P. Tam, H. Li, B. Shen, P.E. Lobie, Growth hormone as a cytokine, Clin. Exp. Pharmacol. Physiol. 26 (1999) 760–764. [17] M.J. Waters, A.J. Brooks, JAK2 activation by growth hormone and other cytokines, Biochem. J. 466 (2015) 1–11. [18] M.J. Waters, A.J. Brooks, Y. Chhabra, A new mechanism for growth hormone receptor activation of JAK2, and implications for related cytokine receptors, JAKSTAT 3 (2014) e29569.
Please cite this article as: M.J. Waters, The growth hormone receptor, Growth Horm. IGF Res. (2015), http://dx.doi.org/10.1016/j.ghir.2015.06.001
