1. Introduction
Growth hormone (GH), also known as somatotropin, is a peptide hormone released from the anterior pituitary somatotroph cells. GH is involved in the promotion of growth, cell division and regeneration [
1,
2], the regulation of metabolism, immune, reproductive, and cardiovascular systems, and the brain [
3,
4]. GH effects are directly mediated through the GH receptor (GHR) and indirectly via insulin-like growth factor 1 (IGF-1). The secretion of GH is pulsatile, occurring under a variety of hormonal influences, such as stimulatory hypothalamic GH-releasing hormone, ghrelin and sex steroids, inhibitory somatostatin, IGF-1, and glucocorticoids. A complex feedback system involving IGF-1, leptin, ghrelin, free fatty acids, and the central nervous system regulates GH secretion. When released, GH binds to GHR in the liver and cartilage, leading to the production of IGF-1, which, through endocrine and paracrine/autocrine mechanisms, stimulates linear bone growth [
5] or initiates other functions [
6].
Notably, GHR plays a key role in the function of the GH–GHR–IGF-1 axis and is also an important factor for individual growth. In this axis, as an essential cytokine, GHR introduces the GH signal into the cell and then regulates the expression of IGFs, thereby regulating individual growth. Hence, the expression level and normal functioning of GHR in cells and tissues directly affects the physiological effects of GH [
7,
8]. Individuals with dysfunctional GHR, experiencing a loss or an abnormality in the GHR response to GH, are extremely short. They also have decreased bone mineral density and increased adiposity, with a greater risk of osteoporosis, lipid disorders, and cardiovascular disease [
9].
Dwarfism is characterized by normal or elevated serum GH and low levels of IGF-1 [
10]. Dwarf phenotypes exist in humans [
3,
7,
11,
12,
13], mice [
14], pigs [
15,
16], cattle, and sheep [
17], and sex-linked dwarfism (SLD) occurs in chicken [
2,
18,
19,
20]. For example, Laron syndrome (LS), also known as growth hormone insensitivity syndrome, and idiopathic short stature (ISS) are autosomal recessive genetic disorders associated with severe postnatal growth failure and is mostly caused by mutation in the human
GHR. Various mutations in the
GHR gene have been reported, including deletion, RNA processing defects, translation stop codons, and missense mutations, which affect ligand binding, GHR dimerization, or signal transduction, which result in the failure to promote body growth [
19,
21,
22]. A similar example is SLD in chicken, which is caused by
GHR gene mutation, including point mutation in a structural gene or regulatory region, splicing site alterations, read frame shift, and complete or partial gene deletion. Moreover, SLD in chickens caused by different mutation types has a wide range of effects on production performance [
18,
19].
3. GHR Polymorphisms and Individual Dwarfism
GH affects growth by binding to GHR in target cells. Given the biological effects exerted by GH through GHR, deletions and mutations in the
GHR gene may affect the expression or function of GHR, after disrupting the effects of the GH/IGF-1 pathway. Consequently, compared to the average individual height and weight in a population, skeletal development is different. Individual dwarfism caused by GHR disorders are LS and ISS in humans [
12,
13,
22,
37,
38,
39], miniature pigs [
15,
16,
40], cattle and sheep [
17], and sex-linked dwarfism (SLD) in chickens [
2,
18,
20].
GHR function abnormalities are primarily related to mutations of the gene structure, including base mutation and fragment deletion. Since the late 1990s, researchers discovered many
GHR mutations, including deletion, nonsense, missense, frameshift, splicing site, and large fragment deletion mutations [
38,
41]. The variations in
GHR gene structure affect the structure and function of its expressed protein, so that GH is unable to bind to the hepatocyte membrane, resulting in growth inhibition, showing a dwarf phenotype.
3.1. GHR Gene Mutations that Causing Aberrant GH–GHR Binding
Mutations in the
GHR gene can alter the ability of
GHR to interact with the GH peptide [
8]. Nearly 30
GHR mutations have been reported to be associated with disturbing the GH–GHR combination (
Table 1 and
Table 2). For example, a homozygous substitution mutation E42K was predicted to impair the binding affinity of GHR to GH in humans, and to be responsible for low serum levels of IGF-1, IGF binding protein (IGFBP)-3, and GH binding protein (GHBP) [
42]. A nonsense mutation in the fourth exon of
GHR (R43X) determines a premature termination in the protein translation process. As a result of the absence of the extracellular portion of the GHR, this patient had undetectable GHBP [
43]. GHR C94S was found to lose its ability to bind to GH [
44]. A 307 G > A substitution in exon 5 of
GHR resulted in the replacement of the amino acid aspartic acid at position 103 with a residue of asparagine (D103N). This substitution involved the highly conserved aspartic acid 103, which could be responsible for damaging GHR functionality [
45]. Mutations identified in humans with short stature, localized in the ECD of
GHR, are responsible for impairing GH binding. Although treating patients with high doses of recombinant human GH (rhGH), produced some IGF-1 for a short time, due to the failure of the compensatory mechanisms, the IGF-1 was insufficient for normal growth, delaying bone age, consequently affecting final height [
45,
46].
For chickens, altering one base (T335C) in
GHR exon 5 resulted in an amino acid changes (F112S) in SLD chicken. Since this site is located proximal to a disulfide bond and is a GH binding site, this substitution reduces GH binding activity on the hepatocyte membrane to less than 10%. This base mutation may cause GH failure or impair the ability of GH to bind to the GHR, reducing or stopping the GH synthesis and secretion of IGF-1 through the GH–GHR–IGF-1 axis, ultimately leading to the growth inhibition phenomenon [
11]. S226I missense mutation in exon 7 was found to be related to SLD chickens because of the deletion of large fragments [
47] (
Table 3).
3.2. GHR Gene Mutations Causing Aberrant GHR Dimerization
Mutations occurring in the dimerization domain (exons 6–8) of GHR affect the formation of the GHR dimer. For instance, heterozygous R179C non-synonymous mutation occurs in
GHR exon 6 [
48], and two other mutations as well. The first is E180X (GAA > TAA), which activates a cryptic splice acceptor resulting in a receptor protein with an 8-amino acid deletion in the extracellular dimerization domain. Although retaining the ability to homodimerize, trafficking to the cell surface was defective [
39]. The second is E180 splice, which affects both GH binding and GHR trafficking, rendering the abnormal GHR nonfunctional [
38].
Furthermore, a deletion of 166 bases of exon 7 resulted in premature termination (M207 fs. X8). This mutation decreases GH binding affinity to the GHR, and would thus be responsible for growth retardation [
49] (
Table 2).
3.3. GHR Mutations Causing GHR Failure of GH Delivery to Downstream Genes
The GHR
2 dimers must be combined by GH sites 1 and 2 before performing its transduction function (
Figure 2a) [
3]. Curiously, mutations localized in the whole sequence of the
GHR gene are associated with the defective pathogenesis of the signal transmission. For example, the affinity of GHR to GH remains normal with GHR H150Q, but signal transduction capacity is inhibited (
Table 1) [
44]. For chicken, the 1.7 kb deletion in the 10 and 3’UTR exon regions is responsible for dwarf chickens [
18]. The mechanism of this mutation is that the lost loci identified by microRNA let-7b can suppress the expression of the
GHR gene. Then, an excess of GHR occurs, which leads to adipose deposition and repressed growth [
2]. Mutations at the cleavage sites resulted in an inability of the transcripts to cleave normally (
Table 3) [
11]. Affecting the critical JAK2-binding Box 1 region of GHR ICD (p.R229H/c.899dupC) can lead to a frameshift and early protein termination, disrupting normal GHR signaling [
23,
63]. In addition, deleting the proline-rich region, or changing the four prolines to alanines, also resulted in GHR deficient signaling [
64]. Furthermore, alternative splicing of the
GHR precursor, mRNA, and truncation of GHR can lead to the synthesis of signaling incompetent GHR. For example, deleting exon 3 GHR represented by a 532-bp fragment terminated the signal transmission in advance [
37,
51]. Truncated GHR protein resulting from exon 8 skipping was directly secreted out of the cell [
65], and truncated GHR missing 184 amino acids and another truncated GHR lacking all but five amino acids of the cytoplasmic domain could not mediate any effects of GH, nor was it internalized [
64]. Truncated GHR 1–277 and 1–279 variants led to a translational frame shift that introduced a stop codon three to four amino acids after the GHR TMD, leading to truncation of the entire cytoplasmic domain (
Table 4) [
6,
66]. Using rhGH or rhIGF-1 to treat the patients with these mutations, although serum IGFBP-3 was normalized or below normal, IGF-1 serum levels were only modestly increased. This means that patients would either lack a response to rhGH or rhIGF-1, which would inhibit downstream GH-induced signaling through the negative feedback loop to the pituitary [
63].
Two truncated GHRs formed a heterodimer (
Figure 2b), but further study is required. Sometimes these truncated GHR produced GHBP [
30]. Reports suggest a predominantly negative effect of truncated variants, as they can form a long–short heterodimer with a full-length GHR, which hampers the dimer signaling (
Figure 2c,d) [
67]. Considering GHR is conserved among different species [
68,
69], this kind of phenomenon can occur with other mammal and avian species.
3.4. Mutations Resulting in GHR Expression Failure
A missense mutation caused by the transformation of adenine to guanine (c.1 A > G) was found in the first codon of exon 2. Given that this substitution involved the translation initiation codon of the protein, the correct expression of the receptor is inhibited [
45]. The sequencing for
GHR exon 5 revealed a TT insertion at nucleotide 422 after codon 122. The insertion resulted in a frameshift introducing a premature termination codon that led to a truncated receptor (
Table 1) [
22]. Additionally, a splice site mutation was located at the donor splice site of exon 2/intron 2 within
GHR, changing the open reading frame of
GHR, resulting in a premature termination codon in exon 3 (
Table 5) [
10]. Also, a subset of the GHR homodimer was cleaved at the cell membrane, releasing the ECD known as the GHBP into the circulation. The mechanism underlying the generation of soluble GHBP likely differs between species. Human
GHR truncation is identical in sequence to full-length
GHR, except for a 26-bp deletion, leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with human
GHR, human truncated
GHR showed a significantly increased capacity to generate soluble GHBP [
70]. Another example is the G679T substitution at the
GHR gene leading to the replacement of S226I. Although the length of the encoded protein did not change, this protein was not expressed on the surface of hepatocytes [
60]. The GHBP is a transcriptional activator in mammalian cells, and this activity occurs in the lower cytokine receptor module. This activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box [
1]. A homozygous 784 G > C transversion induced exon 7 excision and the functional loss of the native intron 7 donor splice site, leading to a frame shift and predicted early protein termination [
62].
Patients with growth hormone insensitivity and without mutations in the
GHR gene coding region should be screened for mutations in the noncoding regions, such as an intronic
GHR mutation within intron 4 (266 + 83 G > T), which generates a 5’ donor splice site to retain 81 intronic nucleotides in the
GHR mRNA. The abnormal splicing event caused early protein termination and undetectable GHBP in the serum [
66]. Intron 6 (A (−1) > G (−1)) substitution lead to the skipping of exon 6 and premature termination of the mRNA message (
Table 5) [
12,
44]. All the mutations in
GHR introns are splice site mutations, which disrupt the expression of GHR.
3.5. GHR Regulates Development of Both Bone and Muscle Fiber
In addition to promoting linear growth, GH plays a crucial role in the regulation of bone and muscle development and metabolism by acting directly through the GHR [
73,
74], or indirectly via hepatic IGF-1 production. Signal transduction errors in the GH–GHR–IGF-1 axis can cause growth failure and changes in body composition. GHR signaling in bone is necessary for establishing radial bone growth and optimizing mineral acquisition during growth [
75].
Longitudinal growth is primarily influenced by the GH–IGF-1 axis, which is a mixed endocrine–paracrine–autocrine system [
76]. Constant manipulation of the GH–IGF-1 axis influences both morphology and mRNA levels of selected genes in the muscle–tendon units of mice. However, only moderate structural changes were observed with up-regulation of the GH–IGF-1 axis; disrupting of the GHR had pronounced effects upon tendon ultrastructure [
77]. GH directly impacts the growth plate to stimulate longitudinal growth, demonstrated by staining the GHR and GHBP located in both the cytoplasm and the nucleus. The localization of GHR/GHBP suggests that, in addition acting on germinal and proliferative cells in young rats, GH also affects early-maturing chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte [
78]. Mutation of the
GHR gene also caused a decrease in the number of muscle fibers, decreasing the myofiber diameter [
79]. GHR signaling in postnatal skeletal muscle was not found to play a significant role in regulating muscle mass or muscle regeneration [
80].
4. Discussion
It is well known that there are two different steps of GH action: at first, GH is directly mediated by GHR to be transducted to IGF1; and then the IGF-1 acts on target cells to exert the physiological effect of GH [
4,
81]. Although GH acts in nearly every tissue of the body, the most known for its growth promoting effect of GH is on cartilage and bone, especially during the adolescent years. Once the JAKs/STATs signal was activated by GH, they were transported into the nucleus to induce increased gene transcription and metabolism to produce IGF-1, which will release into the circulatory system. IGF-1 then binds to its receptor on the cellular surface and activates a JAK-mediated intracellular signaling pathway, which intracellularly phosphorylates various proteins leading to increased metabolism, anabolism, and cellular replication and division [
82].
The effects of GH were mediated by systemic IGF-1, so it should be assumed as a combined effect of both GH and IGF-1. Pituitary GH is the major regulator of liver-produced IGF-I, which is transported via the circulation to peripheral tissues where it acts in an endocrine manner [
83]. However, several evidences support the notion that circulating IGF1 is independent of GH. Glucose, leptin, insulin and proluctin can stimulate the expression of IGF-1 in patients without GH to maintain their normal growth [
84]. Moreover, it was well demonstrated that IGFs regulate bone length of the appendicular skeleton evidenced by changes in chondrocytes of the proliferative and hypertrophic zones of the growth plate. IGFs affect radial bone growth and regulate cortical and trabecular bone properties via their effects on osteoblast, osteocyte, and osteoclast function [
83].
Notably, the functionality of the GH–GHR–IGF-1 axis partially depends on the expression of liver GHR, which determines the amount of IGF1 released from the liver in response to GH [
81]. The loss of GHR leads to a decrease in the synthesis and secretion of IGF-1 [
85]. The net effect of the loss in IGF1 is that leads to a loss of negative feedback on GH, as well as cells may be systemically starved for growth factors and have slower growth or less metabolic activity [
81].
We summarized different mutations of the GHR gene and their possible mechanism relevant to human or chicken’s dwarfism. GHR mutations that occur in the encoding region may cause amino acid changes, thus affecting protein structure and function. Mutations of the splicing sites result in the improper translation of transcripts into biologically active proteins. Deletion mutations lead to a frameshift, affecting the structure and function of proteins. Even in regulatory regions, mutations may affect the temporal and spatial expression patterns of the gene.
Several defects have been reported to be associated with body dwarfism. For instance, the G62V mutation in exon 4 [
53], a heterozygous mutation (V144I) within exon 6 of the
GHR [
13], a dinucleotide deletion on exon 7 of the
GHR gene [
61], and even a GT-repeat microsatellite in the
GHR 5’UTR [
7] can result in dwarfism. However, not all mutations in
GHR cause body dwarfism. For example, mutation of phenylalanine 346 to alanine resulted in a GHR that did not internalize rapidly. However, this mutant GHR was capable of mediating GH-stimulated transcription as well as having metabolic effects [
64]. S325S, L526I [
86], c.–10 T > C (exon 2), G168 (exon 6) and 8 intronic mutations of GHR (662 − 31 C > T, 662 − 30 A > G, 662 − 24delG, 662 − 11delT, 482 + 9 C > T, 828 − 4delG, 919 − 14delT, and 988 + 23delG) [
87] did not induce individual dwarfism. However, the dose individually affects the
GHR mutations causing dwarfism. Various mutations in
GHR genes occur in some individuals, producing additive effects that lead to individual dwarf performance [
87]. For example, the dwarf (
dw) gene in chickens creates a loss of function mutation with a dose effect because the offspring of SLD cocks and normal hens only showed a slight decrease in body weight. From the point of view of traditional genetics, multiple alleles in the dw locus, and the genetic effects of these alleles are also different. When multiple alleles exist at the same time, they may also have additive effects. Similar findings were also found in the pig melanin receptor gene [
88].
Normally, after the GHR–GHR homodimer binding to GH, each GHR molecule is coupled with one or more non-activated kinases through a non-covalent form, including JAK2 and Src family kinases [
34]. However, except for GHR–GHR homodimerization, GHBP–GHBP homodimerization and GHBP–GHR heterodimerization [
63,
89] reactions are pervasive mechanisms that hinder signal transduction [
28]. Diagnosis and treatment of these abnormal phenomena are formidable tasks for researchers.
A transcription of the
GHR gene (GHRG-1, coding 120 amino acids) [
90] and an antisense transcript of
GHR (4337 bp, non-coding RNA) [
91] were only expressed in normal chickens and are considered as new GH regulators. Researchers demonstrated that the dwarf phenotype of the SLD chicken is not only controlled by the full-length
GHR, but it may also be regulated by different transcripts of
GHR. Given the conservativeness of
GHR gene expression in humans and chickens, this process may also contribute to human dwarfism.
Mutations in
GH and
IGF-1 genes are also important when studying body dwarfism, because the mutations of both genes may play roles altering the GH–GHR–IGF-1 axis signaling transduction. GH has no axis of symmetry. Its interaction with GHR is mediated by two distinct asymmetric binding sites with different affinities on GH. Site 1 has high affinity and mediates the first binding step. Mutation of binding site 2, as with the human GH mutant G120R, disrupts the second binding but leaves site 1 binding intact. G120R is a GH antagonist, binding only one GHR and thus fails to signal, and it prevents productive GHR binding by normal GH [
25]. The mechanism by which GH binding converts the inactive pre-dimerized GHR to its active signaling conformation has not been confirmed.