Structure
Somatotropin has three major isoforms. The predominant form is composed out of 191 amino acids and has a molecular weight of 22 kDa.
The primary structure, corresponding to a sequence of amino acids, of the predominant somatotropin is the following :
FPTIPLSRLFQNAMLRAHRLHQLAFDTYEEFEEAYIPKEQKYSFLQAPQASLCFSESIPTPSNREQAQQKSNLQLLRI
SLLLIQSWLEPVGFLRSVFANQLVYGASDSDVYDLLKDLEEGIQTLMGRLEDGSPRTGQAFKGTYAKFDANSHNDDA
LLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF
Somatotropin does not exist as a linear chain of amino acids, it twists and folds on itself, forming the secondary structure. The protein, made up of a single chain, consists of four antiparallel aligned in an up-up-down-down manner [5] [2]. The first helix starts at the 6th amino acid, which is a leucine and ends with the 37th amino acid proline. It is separated from the other three helices after the 37th position. The 38th and 39th amino acids, which are lysine and glutamic acid are spliced out of the protein and therefore disconnects the first helix from the second one. The second helix starts at position 72 till 92, the third from 106 till 128 and the fourth helix from 154 until 184. All helices are ampipathic with strong , especially helix 2 is very hydrophobic. The hydrophobic protein core is usually tigthly packed and any mutations in the hidden positions lead to destablilization [1].
From the secondary structure, we obtain the tertiary structure, which corresponds to the 3D structure adopted by all the alpha helixes. The structural maintenance is stabilised by electrostatic, hydrophobic and polar interactions, and/or covalent interactions with cysteine 53 and cysteine 165 that form a as well as cysteine 182 with cysteine 189 [3]. Cys53 and C165 also link the crossover connection between helices 1 and 2 to helix 4. The other cysteine-pair form a small loop in C-terminus, involved in the receptor binding site 1 with direct contact to the extracellular domain of the GH receptor [4]. It is also requiried for stability, but unlike the first cysteine-pair not essential for biological activity [5]. A disruption of these disulphide bridges drastically reduces the molecule stability, but only if one unpaired cysteine remains intact. This may lead to implifications for diagnosis or treatments of growth disorder [5].
The protein has two different binding sites: both located at the ends of the protein, the N-terminus as well as the C-terminus [6].
The GH consists of two hydrophobic cores, one is composed of Trp104 (hGH-receptor1), Trp169 (hGH-receptor1), Pro61 (hGH), Phe176 (hGH) and Ile176 (hGH). Especially Pro61 is important, it is involved in the formation of the ative conformation of hydrophobic core amino acids and interacting with other hydrophobic core amino acids within 5Å. A mutation in this point leads to a decrease of biological and receptor binding activity. The other one is composed of two pairs of interaction between Trp76 (hGH-receptor1) and Pro48 (hGH) and between Pro106 (hGH-receptor1) and Leu45 (hGH). [1]
The second isoform was found in blood circulation and lost the amino acids 32 till 46 due to alternative splicing of the pre-mRNA and therefore has a molecular weight of 20 kDa [7]. It has a reduced insulin linked activity but is still very similiar to the predominant form, although a quarter of the amino acids in the long loop between helices 1 and 2 was deleted. The loss of these amino acids could be compensated due to the flexibility of the loop region. Through the lack of Lys41, the isoform can not form a saltbridge between hGH and the first receptor. It is possible for the hGH to compensate partially a deficit of 200Å contact surface area through Pro33 and Leu37 in hydrophobic interaction and Arg152 in salt bridge. The third isoform has a molecular weight of 17,5kDa and is formed by alternative splicing of the pre-mRNA. A mutation in the first and sixth basepair leads to a missplicing of the mRNA and loss of exon 3. The GH produced lacks amino acids 32 to 71 and causes isolated GH deficiency type II. The entire connecting loop between helices 1 and 2 and the Cys53 which is required for the first disulphide bridge is missing which leads to unpaired cysteine. Some deleted amino acids are part of a hydrophobic core which is essential to fold the molecule normally. The molecule gets instabil, it can not refold properly. Additionally, the receptor can not bind to binding site 1 which leads to a huge loss in activity. [1] [5]
HGH receptors and interactions
The GH membrane receptor (GHR) is found on many cells and tissues with the exception of the brain, testicles and thymus. It is part of the [class I cytokine receptor family [8]. The nature of this receptor is not fully understood, but it seems that it may be present in different forms due to different post-translational changes that may occur in a single protein.[6]
The hGH receptor is a transmembrane protein consisting of 620 amino acids. It has two extracellular domains which are highly conserved, each containing 7 β-sheets. The protein should theoretically have a molecular mass of 70 kDa considering the amino acid sequence. In fact the actual molecular weight is 100 - 130 kDa. This can be explained due to post-translational modifications such as glycosylation and ubiquitination. Ligand binding increases ubiquitination and possibly has effect on GH receptor internalisation [9].
Binding to the receptor is the first step in the biological action of the hormone.[6] For the binding of a single hGH, two hGH receptors are needed. The hGH binds with its high affinity binding site 1 onto the extracellular domain of the first receptor and with the low affinity binding site 2 onto the extracellular domain of the second receptor [1] [5]. This leads to receptor homodimerization and transmits the signal into the target cell [10]. Additionally, it induces a conformational change in the intrecellular domain of the GHR [11]. The are allosterically coupled, this effect focused among some residues centered around the interaction between Asp116 (hGH) and Trp169 (hGH-receptor2). [1]
The first binding site of the HGH protein contains parts of helices 1 and 4 (amino acids 103-119), as well as parts of the binding loop between helices 1 and 2 (amino acids 41-68). The second binding site contains the and part of the third helix (amino acids 54-74). Major roles hereby play Phenylalanine 1 and Isoleucine 4 (N-terminus) and Aspartic acid 116 [7]. The C-terminal part of the receptor, consisting of the last 165 amino acids, has no effect on GH binding.[6] [1]
Several GH variants with modified N-terminus were explored to learn more about the behaviour of the binding of the receptor molecule. A GH with an extension of Methionin on the N-terminus behave identical to an authentic GH in assays. The small, neutral amino acid without any side chains has no apparent effect on the binding of the receptor. In contrast, a removal of 13 amino acids at the N-terminus results in reduced binding activity to somatogenic receptors and decreased biological activity, because the amino acids 1 to 16 are involved in the binding site 2. But the other part of the binding site (with Trp103, Asp116 and Glu119 from helix 3) of the hGH molecule are intact and available for binding of the receptor. The N-terminus is required for full biological activity, however, additions still deletions can not completly abolish the functions of the molecule. [1]
The hormone-binding extracellular domain consists of 250 amino acids, including several cysteine residues which are conserved and can form disulphide bridges.The intracellular domain of the receptor is made up of 350 amino acids, it represents the least conserved region and is made up of 10 tyrosine residues likely to be phosphorylated by tyrosine kinase (JAK2), during the formation of the GH-receptor complex. A reaction cascade involving kinase enzymes is then activated, allowing the expression of certain genes coding for proteins or not, necessary for biological activity [12]. It therefore controls the expression of certain genes such as the gene coding for the IGF-1 factor. The liver and adipose tissue being important targets for GH, it therefore contributes to metabolic homeostasis.[6] [13] The intercellular domain containing of two box regions. The proline rich first one and an acidic, hydrophobic second one which is connected to receptor internalizing mechanisms [14].
Dieseas and treatments
Many diseases can be due to a dysfunction in the secretion of GH.
Dwarfism is characterized by a lack of GH. The reasons for this deficiency can be explained by many fact, as welle as : a dysfunction during foetal development,The inability of somatotropic cells to synthesize GH, an abnormal structure of the protein, making the connection to the receiver more difficult, geneics mutation ... These factors are then likely to lead to a slowdown in growth, cell, tissue, and bone development.[8]
Gigantism is characterised by the presence of a high level of GH or IGF-1. This pathology is most often due to an adenoma of the pituitary cells, responsible for the production of the hormone GHRH, which then stimulates the cells to produce GH in large quantities. It can also be explained by the fact that tissues that do not normally produce GH have tumour cells capable of producing GH. Acromegaly is when this excess of hormone occurs after puberty.[8]
Until 1985, injections of GH were carried out for people suffering from dwarfism. As it could only be obtained by extraction from the pituitary glands of dead people, it could only be extracted in small quantities, so resources were limited.[9] Also this therapeutic treatment has been stopped in many countries, due to the possible contamination of the hormone by prions, which can cause serious diseases such as Creutzfeldt-Jakob disease.[8] As a result, biosynthetic synthesis of the hormone is carried out by developing recombinant proteins of GH or IGF-1.[9]
Affected by gigantism pathology, individuals may have therapeutic radiation and therapeutic drug treatment or synthesis of inhibitors such as somatostatin,[8] which act as GH antagonists by binding to the receptor, thus preventing the GH to perform its functions.
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Sami AJ. Structure-function relation of somatotropin with reference to molecular modeling. Curr Protein Pept Sci. 2007 Jun;8(3):283-92. doi: 10.2174/138920307780831820. PMID:17584122 doi:http://dx.doi.org/10.2174/138920307780831820
- ↑ Barsh GS, Seeburg PH, Gelinas RE. The human growth hormone gene family: structure and evolution of the chromosomal locus. Nucleic Acids Res. 1983 Jun 25;11(12):3939-58. doi: 10.1093/nar/11.12.3939. PMID:6306568 doi:http://dx.doi.org/10.1093/nar/11.12.3939
- ↑ Reh CS, Geffner ME. Somatotropin in the treatment of growth hormone deficiency and Turner syndrome in pediatric patients: a review. Clin Pharmacol. 2010;2:111-22. doi: 10.2147/CPAA.S6525. Epub 2010 Jun 1. PMID:22291494 doi:http://dx.doi.org/10.2147/CPAA.S6525
- ↑ Utiger, R. D. and other Encyclopedia Britannica Contributors (1998), Insulin-like growth factor. Science, Chemistry.
- ↑ 5.0 5.1 5.2 5.3 Junnila RK, Kopchick JJ. Significance of the disulphide bonds of human growth hormone. Endokrynol Pol. 2013;64(4):300-5. doi: 10.5603/ep.2013.0009. PMID:24002958 doi:http://dx.doi.org/10.5603/ep.2013.0009
- ↑ 6.0 6.1 6.2 6.3 Le Cam, A. (1993), Mode d’action de l’hormone de croissance. médecine/sciences, 12:1352-61.[1]
- ↑ Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA. Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science. 1991 Nov 8;254(5033):821-5. doi: 10.1126/science.1948064. PMID:1948064 doi:http://dx.doi.org/10.1126/science.1948064
- ↑ 8.0 8.1 8.2 8.3 Utiger, R.D. and other Encyclopedia Britannica Contributors (1998), Growth hormone. Life cycle, processes & properties encyclopedia articles.
- ↑ 9.0 9.1 Dr. Abdi, M., Département de Génétique Moléculaire et Appliquée, Université des Sciences et de la Technologie d’Oran Mohamed BOUDIAF.
