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II THE GROWTH HORMONE RECEPTOR
A The Growth Hormone Receptor Superfamily
The GH receptor locus maps to the proximal short arm of human chromosome 5, region p13.1-p12 (Barton et al., 1989). The cDNA for the human GH receptor encodes a 638 amino acid protein with single extracellular, transmembrane, and cytoplasmic domains. Numerous receptors, including the GH receptor, were shown to have limited amino acid homology in a region of the extracellular domain, prompting the classification of a new cytokine receptor superfamily, of which the GH receptor was the first identified member (Cosman et al., 1990; Due et al., 1995). Other members of the class I cytokine receptor superfamily include prolactin, leptin, erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), neurotrophic factor (CNTF), thrombopoietin, and interleukins (IL) 2–7, IL-9, IL-11, and IL-12. A second class of more distantly related cytokine receptors has been classified and includes the interferon α, β, and γ receptors and the IL-10 receptor (Cosman et al., 1990; Ihle et al., 1995). Characteristics of the members of the class I receptor superfamily include the following (Bazan, 1989; Patthy, 1990): (i) presence of a single putative transmembrane domain, (ii) limited amino acid homology (14–44%) in a region spanning approximately 210 amino acids in the extracellular domain, (iii) three conserved pairs of cysteine residues in the extracellular domain and a conserved tryptophan residue adjacent to the second cysteine in the N-terminal fibronectin domain, (iv) a WSXWS (Trp, Ser, Any, Trp, Ser)-like motif in the juxta-membrane fibronectin domain (YXXFS in the mammalian GH receptor) (Waters, 1997), (v) the absence of a canonical tyrosine kinase consensus sequence, and (vi) two proline-rich motifs (termed box 1 and box 2) in the membrane-proximal region of the cytoplasmic domain (Fig. 1).
FIGURE 1. Schematic structure of the GH receptor. Features of the GH receptor include a WSXWS-like motif, three conserved pairs of cysteine (C) residues and several N-glycosylation sites (N) in the extracellular domain, and a transmembrane domain, as well as two proline-rich boxes and numerous tyrosine (Y) residues within the intracellular domain.
A single arginine at residue 43 of the extracellular domain determines the specificity of human GH (hGH) to bind to the hGH receptor (Souza et al., 1995). Box 1 is 8 amino acid residues in length and is located within 20 residues of the transmembrane domain. Box 1 is one site of association of JAK2 with the GH receptor and is critical for many GH-stimulated cellular functions (Goujon et al., 1994; VanderKuur et al., 1994; Frank et al., 1995). Box 2 comprises a cluster of approximately 15 hydrophobic and acidic amino acid residues and is located 30 residues distal to box 1. Deletion or mutation of these boxes abrogates both JAK activation and subsequent signal transduction pathways responsible for GH-stimulated cell proliferation (Dinerstein et al., 1995; Tanner et al., 1995).
The growth hormone-binding protein (GHBP) is a soluble, circulating form of the GH receptor (Herington et al., 1986). It is differentially secreted among species and is produced by two different mechanisms: by alternative splicing in mice and rats (Baumbach et al., 1989), resulting in the addition of a unique carboxyl-terminal hydrophilic “tail” sequence, and by proteolysis of the membrane-bound form of the receptor in other species (Sotiropoulos et al., 1993). At least one of the proteases involved in this cleavage is the metalloprotease “sheddase” ADAM-17 (Zhang et al., 2000), which has been shown to cleave the GH receptor at a site eight residues from the membrane in the proximal extracellular domain (Wang et al., 2002). Some C-terminally truncated, but still membrane-anchored isoforms of the GH receptor have been described in humans (Dastot et al., 1996; Ross et al., 1997) and these appear to have a greater susceptibility to juxta-membrane proteolysis. The GHBP is capable of binding GH with high affinity in the circulation and in the extracellular environment. It has no known direct biological effect on cells nor any GH signaling capacity; however, it has been shown to significantly modulate (inhibit) cellular GH action (Lim et al., 1990) via one of two presumed mechanisms: (i) direct sequestration of GH away from the cell membrane-bound GH receptor and (ii) heterodimerization with the full-length, wild-type receptor on the cell surface (Herington, 1994). As indicated in Sections II,B and III,A, dimerization of two full-length receptor molecules is required to generate normal GH receptor signaling within the cell and hence the truncated GHBP can act as a “dominant-negative” form of the receptor (Ross et al., 1997). Studies by Graichen et al. (2003) have indicated that endogenous GHBP can be actively translocated to the cell nucleus and can act as a STAT5-mediated transcriptional enhancer, thereby raising the possibility that it may play a direct transcriptional role via interaction with nuclear proteins or DNA.
B Receptor Dimerization
The use of X-ray crystallography has enabled visualization of the three-dimensional structure of the GH receptor extracellular domain and the regions that interact with GH (DeVos et al., 1992). A single GH molecule binds to two molecules of the GH receptor, leading to receptor homodimerization. GH binding to the two GH receptors is thought to be sequential. The initial step is “high-affinity” (site 1) binding of GH to one GH receptor. A different face of GH then contacts the second GH receptor via “lower affinity” (site 2) binding. The contact with hGH at site 2 and a dimerization interface of approximately 500 Å between the two extracellular domains of the receptor stabilize the binding of the second receptor to the complex (Wells, 1996). Dimerization of the GH receptor does not produce major conformational changes in the extracellular domain (Clackson et al., 1998). At present, the structure of the cytoplasmic domain of the GH receptor is not well understood.
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B Structure of the Growth Hormone Receptor
The structure of the intracellular domain of the growth hormone receptor as a hormone-bound complex containing two receptor extracellular domains and one growth hormone molecule has been elucidated by X-ray crystallography (Fig. 2) (33). The receptor can be seen to contain two mutually perpendicular domains of approximately 100 amino acids. These are equivalent to the SD-100 motifs modeled by Bazan (27). Both the C-terminal and N-terminal SD-100 domains contain seven β strands that form a sandwich of two antiparallel β sheets, one with four strands and one with three. The two distinct domains are linked by a short four-residue segment. The N-terminal domain contains three disulfide bonds, one of which cross-links the two β sheets.
Fig. 2. Structure of the growth hormone receptor. Ribbon diagram of the extracellular domain of the human growth hormone receptor/human growth hormone 2:1 complex. The “WSXWS” box and cysteine bridges are shown in ball and stick format. (Data derived from Ref. 38a.)
The crystal structure also shows that growth hormone possesses two unique receptor-binding sites. Site 1 has much higher affinity than site 2 for the receptor. Virtually the same receptor residues are involved in binding both site 1 and site 2. However, there is a clear element of asymmetry because the two receptor molecules are shifted from perfect twofold symmetry by about 10 Å with respect to each other. The area buried between growth hormone site 1 and the receptor (2 700 Å2) is considerably larger than that observed in the growth hormone site 2-receptor interaction (1500 Å2). This difference is compensated for by the additional surface that is buried between the two C-terminal domains of the receptors (900 Å2). Interestingly, the interaction between the receptors at the dimer interface is much more extensive than expected. Such close interaction (2-3 Å) is not observed in the interferon-7 receptor crystal structure (34). Receptor-receptor interaction appears to contribute significantly to the overall stability of the receptor extracellular domain-growth hormone complex.
Although strict sequence alignment with other receptors with WSXWS motifs indicates that the human growth hormone receptor WSXWS ranges from Tyr-222 to Ser-226 (YGEFS), structural studies performed in our laboratory suggest a more significant role for residues Lys-179, Trp-186, Arg-211, Phe-225, Arg-213, Tyr-222, and Lys-215. This motif might serve to stabilize the C-terminal SD-100 domain, which lacks intramolecular disulfide bridges. Because the receptor binding sites lie close to the “hinge” region between the two SD-100 domains, it is tempting to speculate that stabilization of the angle between the two domains may be one of the events leading to receptor activation (Fig. 1). Unfortunately, the structure of the free, unbound receptor is not yet available. Thus it is not known whether the receptor conformation changes following growth hormone binding (see Note Added in Proof).
Several lines of evidence, in addition to the growth hormone receptor extracellular domain crystal structure, indicate that ligand-driven receptor dimerization is the key event leading to transduction of cytokine signals across the cell membrane. A 2:1 complex between the human growth hormone extracellular domain and human growth-hormone is observed both in solution and following crystallization (35). A human growth hormone single-site mutant Gly-120 to Arg (G120R), deficient in receptor binding site 2, inhibits receptor dimerization in vitro and inhibits signal transduction in vivo (36–38). G120R human growth hormone crystallizes as a 1:1 complex with the extracellular domain of the human growth hormone receptor (38a).
The response to growth hormone in several model systems shows bipha-sic bell-shaped kinetics. Moderate levels of hormone stimulate signaling by inducing the formation of the active 2:1 receptor hormone complex whereas an excess of hormone leads to the formation of 1:1 receptor complexes, which inhibits signaling (39). Prolactin receptor antibodies mimic the action of prolactin on Nb2 pre-T lymphocytes by stimulating the formation of receptor dimers (40). Finally, insertion of a cysteine in the membrane-proximal region of the extracellular region of the erythropoietin receptor creates a con-stitutively dimerized and hence active receptor (41). The distribution of the growth hormone receptor on the cell surface is not clear, although there is some evidence for the presence of other membrane-bound receptors in specialized regions of the cell membrane. Thus it is a matter of speculation as to whether receptor monomers are brought together following ligand binding to form dimers by lateral diffusion, or whether preformed dimers are stabilized as a result of hormone binding. There is evidence that a cysteine bridge between receptors may stabilize the growth hormone receptor dimer following ligand binding (42).
The intracellular domain of the growth hormone receptor is still virtually a black box. Nothing is known about its three-dimensional structure. The only region with a defined role in signaling is the proline-rich box 1 region, which binds the cytoplasmic tyrosine kinase Janus kinase 2 (JAK 2). Several other molecules involved in cytokine signaling also bind to the growth hormone receptor. These include insulin receptor substrate 1 (IRS-1), Grb-2, and SHC (43,44). The exact location of the binding sites for these proteins remains to be established.
The intracellular domain of the growth hormone receptor also contains 10 tyrosine residues. A role in activation of protein synthesis and lipogenesis has been ascribed to the two tyrosines closest to the membrane (45). However, the phosphorylation status of the other tyrosines following ligand binding to the receptor is unclear. Indeed, it is still not known which tyrosines are exposed to the cytoplasm. A potential role of the other tyrosines in activation of gene transcription is discussed in Section III,B.
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I INTRODUCTION
In 1993, the growth hormone (GH) receptor was first observed to bind to the tyrosine kinase, Janus kinase 2 (JAK2). GH increased the affinity of JAK2 for the GH receptor, potently activated JAK2, and stimulated the phosphorylation of tyrosine residues within JAK2 and the cytoplasmic domain of the GH receptor (Argetsinger et al., 1993). In the intervening decade, numerous signaling molecules have been identified that are activated in response to GH. Activation of these molecules results in various biochemical and biological outcomes, which are rapidly being elucidated and, ultimately, provide a basis for understanding GH action.
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Growth Hormone (hGH)
Within the cytokine superfamily, the growth hormone (GH)–receptor complex is undoubtedly the most well-characterized ligand–receptor system, and may thus be viewed as a paradigm of structure–function studies. hGH is a monomeric protein, and binds the hGH receptor in a 1 : 2 ratio via two non-identical sites, called sites 1 and 2. Several crystal structures of hGH in complex with the GH receptor have been reported, revealing in atomic detail the basis of cytokine–receptor recognition, i.e. the “structural epitope” [3, 8]. Complementing this, mutagenesis studies have explored the binding determinants for both receptor sites on hGH, defining the “functional epitopes” [2, 6, 9–11].
Variants of hGH exhibiting improved binding affinity to the hGH receptor have been identified through an extensive affinity maturation approach [9], and one such “super-binding” variant, hGHv, encompassing a total of 15 single-substitutions, was demonstrated to bind with a 400-fold enhanced affinity to the hGH receptor. Still, the increased affinity of this and other hGH variants did not in general translate into higher activities (lower EC50 values) [11]. Models of receptor activation have indicated that for high-affinity GH analogs, the cytokine–receptor complex life-time – and in turn signaling efficiency – is independent of binding affinity, but rather limited by the rate of internalization [12], thus illustrating that affinity and activity are not necessarily directly linked. However, affinity maturation of porcine GH led, in contrast to the human GH, to a five-fold higher potency. It was suggested that the increased potency was due to a more efficient activation of the GH receptor stemming from an optimized alignment of the two receptor chains by the mutant compared to wild-type porcine GH [13].
Interestingly, structural characterization of the high-affinity hGH variant revealed significant differences between the receptor interaction surfaces of hGH and hGHv. The two ligands interact through common but, importantly, distinct epitopes, revealing a high degree of plasticity at the site which through different local conformations can interact with high affinity to the hGH receptor. The wild-type cytokine thus represents but one such interaction surface, the high-affinity variant another [14].
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Growth Hormone, IGF-I, Binding Proteins, and Receptors
Growth Hormone
Growth hormone, or somatotropin, is a 191–amino acid protein that contains two disulfide bonds that give it a three-dimensional conformation that is necessary for its biological activity. Release of the growth hormone is triggered by GHRH and is inhibited by somatostatin. The growth hormone plays a negative feedback role in the somatotropic axis, feeding back at the level of the GHRH neuron in the hypothalamus to inhibit GHRH mRNA levels and synthesis and stimulating somatostatin gene expression and release. Not only does the regulation of growth hormone involve the balance between GHRH and somatostatin, but it also involves other neurotransmitters and neuroactive substances, such as dopamine, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, TRH, galanin, neuropeptide Y, motilin, and interleukins. In addition, other hormones, such as the thyroid hormone, glucocorticoids, sex steroid hormones, and metabolic fuels, can influence the release of growth hormone, highlighting the interwoven nature of the different neuroendocrine systems.
The growth hormone is released into the general circulation, where approximately 50% of this molecule is transported in association with a growth hormone–binding protein that is structurally similar to the extracellular domain of the growth hormone receptor. This molecule and the growth hormone receptor (GH-R) are alternative splice products of the same precursor RNA: one encoding the growth hormone–binding protein and the other the GH-R.
IGF-I
A major target for the growth hormone is the IGF-I system. IGF-I is a growth factor that exerts effects on neurite outgrowth and nervous system development. In addition, it is an important mediator of the somatotropic axis and, in fact, was originally referred to as somatomedin. Both GH and IGF-I individually have similar effects on somatic growth and development; however, the two hormones act much more efficiently in combination. In addition, it appears that the growth-promoting effects of IGF-I are particularly important in the central nervous system. This is consistent with the observation that along with its production by the liver, IGF-I is also produced in the brain, and it is probably this central IGF-I that is responsible for many of its neurotrophic effects.
Like the growth hormone, IGF-I plays a feedback role in the inhibition of GHRH biosynthesis (Fig. 38.5). This may involve central and not peripheral IGF-I, as most animal studies have demonstrated that only centrally but not peripherally administered IGF-I has this effect. At the level of the pituitary, IGF-I appears to be the major regulator of negative feedback of growth hormone synthesis and release. Somatotropes contain IGF-I receptors, and IGF-I inhibits growth hormone gene expression and release under basal and GHRH-stimulated conditions. IGF-I can also act as a negative feedback regulator of GHRH neurons, as it can cross the blood–brain barrier to a limited extent. Moreover, IGF-I is synthesized in the central nervous system, including the hypothalamus, and therefore local IGF-I can exert an influence on GHRH neurons. It also has been shown that IGF-I can stimulate somatostatin release, albeit at higher doses than those necessary to inhibit GHRH release, suggesting that IGF-I may be more involved in the suppression of GHRH than the enhancement of somatostatin levels.
IGF-I in the circulation binds to several binding proteins, the major one being IGF-I binding protein 3 (IGFBP-3). Free IGF-I is more biologically active than bound IGF-I, and therefore IGFBP-3 levels are an important regulator of the actions of IGF-I.
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Growth Hormone
Growth hormone is secreted by the anterior pituitary gland largely during SWS and mediates the effects of IL-1 on sleep [59]. Growth hormone may be administered exogenously or stimulated endogenously with prolonged wakefulness [60], exercise [61], and growth hormone releasing hormone (ghrelin). Growth hormone releasing hormone stimulates GABAergic neurons in the preoptic area of the hypothalamus [16]. Accordingly, increased growth hormone leads to robust increases in SWS and SWA [62], specifically in men, with either no change in spindle activity [62] or a reduction in spindle activity [63].
Growth hormones play a well-established role in protein synthesis and adult neurogenesis [64,65]. Growth hormone therapy is associated with improved cognitive function in patients with low growth hormone levels and may be an effective treatment for cognitive impairment [64]. SWS may facilitate growth hormone release and thereby influence synaptic plasticity through growth hormone receptors involved in potentiation in the hippocampus and elsewhere in the brain [64,65]. However, SWS increases improve memory [66], independently from whether growth hormone increases or not, suggesting that cognitive benefits from SWS do not depend on growth hormone. It is still possible, however, that growth hormone by itself increases SWA, leading to large-scale neural synchrony and synaptic scaling with the potential to enhance cognitive function.
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A Endocrine, Paracrine, and Autocrine Sources of Growth Hormone and IGF-I Act on the Brain
Although the brain, with the exception of the hypothalamus, has not typically been considered a target for growth hormone action, autoradiography has demonstrated the expression of growth hormone receptors in the choroid plexus, hippocampus, pituitary, and spinal cord in rodents, with reduced receptor density observed in the cortex (Zhai et al., 1994). Growth hormone receptors have also been observed in human choroid plexus (Lai et al., 1991), and growth hormone receptor mRNA has been observed in human brain stem (Mercado et al., 1994) and astrocytic tumors (Castro et al., 2000). While the main source of growth hormone is the anterior pituitary gland, brain tissues appear to produce small amounts of the hormone that can act at receptors in local tissues. Therefore, paracrine growth hormone action coexists with pituitary-derived circulating growth hormone, which may be transported across the blood–brain barrier via receptor-mediated transcytosis (Coculescu, 1999). Using coronary artery perfusion techniques in rats, Reinhardt and Bondy (1994) demonstrated that liver-derived circulating IGF-I crosses the blood–brain barrier and can then bind IGF-I receptors believed to be expressed throughout the brain, although concentrated in the superficial and deep cortical layers, olfactory bulb, amygdala, thalamic nuclei, and hippocampus (Araujo et al., 1989; Reinhardt and Bondy, 1994; Lesniak et al., 1988; Dore et al., 1997b; Breese et al., 1991; Bondy et al., 1992). Substantial evidence also shows that brain tissue produces IGF-I, which can also act in a paracrine or autocrine fashion (Schneider et al., 2003).
Brain expression of growth hormone and IGF-I, as well as their receptors, is modulated by a variety of factors outside the growth hormone/IGF-I axis. For instance, Fujikawa et al. (2000) demonstrated that, in response to acute stress, growth hormone receptor mRNA expression paralleled increases in plasma glucocorticoid levels and mRNA levels of glucocorticoid and mineralocorticoid receptors in rat dentate gyrus (Fujikawa et al., 2000). Furthermore, transgenic mice overexpressing the cytokine tumor necrosis factor (TNF-α) demonstrate decreased abundance of IGF-I in the cerebellum, cerebral cortex, and diencephalon. Increased expression of IGFBP-3 and decreased levels of IGFBP-5 were also observed in cerebellar tissue of these animals when compared to wild type (Ye et al., 2003). Several investigators have hypothesized that the developmental expression of some IGFBPs may affect IGF-I synthesis in the brain. For instance, spatiotemporal correlations in the expression of IGFBP-2 and -5 mRNA with IGF-I mRNA in the developing rat brain have been reported (Lee et al., 1992; Bondy and Lee, 1993). In summary, the growth hormone/IGF-I axis is important to structures throughout the brain, and expression of its components is regulated by one another and regulates a number of other endocrine and non-endocrine factors.
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83.2.4.13 Growth Hormone Secretagogus Receptor (GHSR) Defects
GHSR (OMIM 601898) is a G-protein-coupled receptor highly expressed in the pituitary and hypothalamus. First recognized as a receptor for exogenous stimulators of growth hormone secretion, its endogenous ligand Ghrelin (GHRL, OMIM 605353) was identified in 1999. Ghrelin has pleiotropic effects on many body systems and is an orexigenic hormone, but it is also known to release GH, suggesting that it or its receptor GHSR might be involved in short stature. In 2006, Pantel et al. identified a GHSR mutation, A204E, in two unrelated families with apparent idiopathic short stature (ISS). The pedigrees suggested autosomal dominant inheritance with more severe expression in one patient who was homozygous. This amino acid is conserved across all species studied so far and the substitution indicates a significant change in charge. In vitro studies showed that the mutation selectively impaired the constitutive activity of GHSR (97). Subsequently, the same group identified a patient who was a compound heterozygote for two mutations in GHSR. The heterozygous parents were unaffected suggesting autosomal recessive inheritance. The patient had laboratory studies consistent with IGHD and responded well to GH. The mutations also showed reduced constitutive activity of the receptor (98).
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Growth hormone pathway
The growth hormone (GH) pathway is composed of a series of interdependent genes whose products are required for normal growth (see Fig. 1). The GH pathway genes include ligands [GH, insulin-like growth factor-I (IGF-I)], transcription factors [Prophet of PIT1 (PROP1) and PIT1], agonists [GH-releasing hormone (GHRH)], antagonists (somatostatin or GH1F), and receptors [GH-releasing hormone receptor (GHRHR) and the GH receptor (GHR)]. These genes are expressed in different organs and tissues including the hypothalamus, pituitary, liver, and bone (see Fig. 1). The effective and regulated expression of the GH pathway is essential for growth in stature as well as homeostasis of carbohydrate, protein, and fat metabolism. Figure 1 shows the GH pathway including its interdependent genes that are required to produce transcription factors, ligands such as GH, receptors such as GHR, and other gene products needed for normal growth. Known gene defects in the GH pathway that cause disease include the following: (1) in combined pituitary hormone deficiency (CPHD), defects in the genes HESX1, LHX3, PROP1, and PIT1 are found; (2) in isolated GH deficiency (IGHD), there are defects in GH1 and GHRHR; and (3) in GH resistance, there are defects in the genes GHR and IGF-I.
Figure 1. The growth hormone (GH) biosynthetic pathway (for details, see text).
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1. GROWTH HORMONE BINDING PROTEIN (GHBP)
Growth hormone exerts a multitude of biological effects on various tissues through the GH receptor. Regulation of GH bioactivity occurs at several pre- and postreceptor levels. Growth hormone binding protein (GHBP) is a plasma binding protein identical to the extracellular domain of the tissue GH receptor [27]. GHBP binds exclusively to GH, and most, if not all, serum GH is bound to this carrier protein [29]. Measurements of GH binding protein in serum are relatively stable and reflect the endogenous status of the GH receptor in responsive tissues [28]. With advanced age, GHBP concentrations increase substantially [27].
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