Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

A novel peptide antagonist of the human growth hormone receptor.

Excess circulating human growth hormone (hGH) in vivo is linked to metabolic and growth disorders such as cancer, diabetes, and acromegaly. Consequently, there is considerable interest in developing antagonists of hGH action. Here, we present the design, synthesis, and characterization of a 16-residue peptide (site 1-binding helix [S1H]) that inhibits hGH-mediated STAT5 phosphorylation in cultured cells. S1H was designed as a direct sequence mimetic of the site 1 mini-helix (residues 36-51) of wild-type hGH and acts by inhibiting the interaction of hGH with the human growth hormone receptor (hGHR). In vitro studies indicated that S1H is stable in human serum and can adopt an α-helix in solution. Our results also show that S1H mitigates phosphorylation of STAT5 in cells co-treated with hGH, reducing intracellular STAT5 phosphorylation levels to those observed in untreated controls. Furthermore, S1H was found to attenuate the activity of the hGHR and the human prolactin receptor, suggesting that this peptide acts as an antagonist of both lactogenic and somatotrophic hGH actions. Finally, we used alanine scanning to determine how discrete amino acids within the S1H sequence contribute to its structural organization and biological activity. We observed a strong correlation between helical propensity and inhibitory effect, indicating that S1H-mediated antagonism of the hGHR is largely dependent on the ability for S1H to adopt an α-helix. Taken together, these results show that S1H not only acts as a novel peptide-based antagonist of the hGHR but can also be applied as a chemical tool to study the molecular nature of hGH-hGHR interactions.

Identification, characterization, and expressions profile analysis of growth hormone receptors (GHR1 and GHR2) in Hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus polyphekadion ♂).

Growth hormone is an essential hormone that plays essential roles in growth, metabolism, cellular differentiation, immunity and reproduction in fish, by means of the growth hormone receptors. The encoding cDNA growth hormone receptors (GHR1 and GHR2) were cloned and characterized from Hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus polyphekadion♂). Sequence analysis of the cloned GHR1 was observed as containing 2176, which comprised an ORF of 1842 bp, 5 UTR of 6 bp and 3 UTR of 328 bp, with 612 amino acids encoding proteins, while GHR2 was observed as containing 1824 bp that encompassed an ORF of 708 bp, 5 UTR of 48 bp and 3 UTR of 1068 bp with 235 amino acids encoding proteins. Relative mRNA expression of GHR1 and GHR2 in the liver and muscle was found to be highest respectively. Our findings provide vital statistics of GHRs likely to play a significant role in the growth of the fish.

Structural and energetic study of cation-π-cation interactions in proteins.

Cation-π interactions of aromatic rings and positively charged groups are among the most important interactions in structural biology. The role and energetic characteristics of these interactions are well established. However, the occurrence of cation-π-cation interactions is an unexpected motif, which raises intriguing questions about its functional role in proteins. We present a statistical analysis of the occurrence, composition and geometrical preferences of cation-π-cation interactions identified in a set of non-redundant protein structures taken from the Protein Data Bank. Our results demonstrate that this structural motif is observed at a small, albeit non-negligible frequency in proteins, and suggest a preference to establish cation-π-cation motifs with Trp, followed by Tyr and Phe. Furthermore, we have found that cation-π-cation interactions tend to be highly conserved, which supports their structural or functional role. Finally, we have performed an energetic analysis of a representative subset of cation-π-cation complexes combining quantum-chemical and continuum solvation calculations. Our results point out that the protein environment can strongly screen the cation-cation repulsion, leading to an attractive interaction in 64% of the complexes analyzed. Together with the high degree of conservation observed, these results suggest a potential stabilizing role in the protein fold, as demonstrated recently for a miniature protein (Craven et al., J. Am. Chem. Soc. 2016, 138, 1543). From a computational point of view, the significant contribution of non-additive three-body terms challenges the suitability of standard additive force fields for describing cation-π-cation motifs in molecular simulations.

The role of protein "Stability patches" in molecular recognition: A case study of the human growth hormone-receptor complex.

Dynamic characteristics of protein surfaces are among the factors determining their functional properties, including their potential participation in protein-protein interactions. The presence of clusters of static residues-"stability patches" (SPs)-is a characteristic of protein surfaces involved in intermolecular recognition. The mechanism, by with SPs facilitate molecular recognition, however, remains unclear. Analyzing the surface dynamic properties of the growth hormone and of its high-affinity variant we demonstrated that reshaping of the SPs landscape may be among the factors accountable for the improved affinity of this variant to the receptor. We hypothesized that SPs facilitate molecular recognition by moderating the conformational entropy of the unbound state, diminishing enthalpy-entropy compensation upon binding, and by augmenting the favorable entropy of desolvation. SPs mapping emerges as a valuable tool for investigating the structural basis of the stability of protein complexes and for rationalizing experimental approaches, such as affinity maturation, aimed at improving it.

Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes.

Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

Identification of Polymorphisms in the Rabbit Growth Hormone Receptor (GHR) Gene and Association with Finishing Weight in a Commercial Meat Rabbit Line.

A shortcut to identify DNA markers associated with economic traits is to use a candidate gene approach that is still useful in livestock species in which molecular tools and resources are not advanced or not well developed. Mutations in the growth hormone receptor (GHR) gene associated with production traits have been already described in several livestock species. For this reason GHR could be an interesting candidate gene in the rabbit. In this study we re-sequenced all exons and non-coding regions of the rabbit GHR gene in a panel of 10 different rabbits and identified 10 single nucleotide polymorphisms (SNPs). One of them (g.63453192C>G or c.106C>G), located in exon 3 was a missense mutation (p.L36V) substituting an amino acid in a highly conserved position across all mammals. This mutation was genotyped in 297 performance tested rabbits of a meat male line and association analysis showed that the investigated SNP was associated with weight at 70 days (P < 0.05). The most frequent genotype (GG) was in animals with higher weight at this age, suggesting that the high directional selection pressure toward this trait since the constitution of the genotyped line might have contributed to shape allele frequencies at this polymorphic site.

[Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies].

Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy.

A ligand-receptor fusion of growth hormone forms a dimer and is a potent long-acting agonist.

Cytokine hormones have a short plasma half-life and require frequent administration. For example, growth hormone replacement involves daily injections. In common with other cytokines, the extracellular domain of the growth hormone receptor circulates as a binding protein, which naturally prolongs the biological half-life of growth hormone. Here we have studied the biological actions of a ligand-receptor fusion of growth hormone and the extracellular domain of its receptor. The genetically engineered ligand-receptor fusion protein was purified from mammalian cell culture. In rats, the ligand-receptor fusion had a 300-times reduced clearance as compared to native growth hormone, and a single injection promoted growth for 10 d, far exceeding the growth seen after administration of native growth hormone. The ligand-receptor fusion forms a reciprocal, head-to-tail dimer that provides a reservoir of inactive hormone similar to the natural reservoir of growth hormone and its binding protein. In conclusion, a ligand-receptor fusion of cytokine to its extracellular receptor generates a potent, long-acting agonist with exceptionally slow absorption and elimination. This approach could be easily applied to other cytokines.

Ubc13 and COOH terminus of Hsp70-interacting protein (CHIP) are required for growth hormone receptor endocytosis.

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(ßTrCP) (Skp Cullin F-box). Despite a specific interaction between ß-transducin repeat-containing protein (ßTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(ßTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to ßTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.

Structural and functional basis for hormone binding and receptor oligomerization.

Most single-pass transmembrane receptors undergo a change in oligomeric state upon hormone binding. Recent mutational, biophysical and structural studies of the human growth hormone and tumor necrosis factor receptor complexes have revealed much about the mechanisms and molecular bases for binding and oligomerization. Principles learned from these examples and others should apply to many other hormone-receptor complexes.

Characterization of the GHR gene genetic variation in Chinese indigenous goat breeds.

The aim of the present work was to investigate single nucleotide polymorphism (SNP) of growth hormone receptor (GHR) gene exon 10, characterize the genetic variation in three Chinese indigenous goat breeds, and search for its potential association with cashmere traits. In this study, a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocol has been developed for rapid genotyping of the GHR gene in goats. One hundred seventy-eight goats from Liaoning Cashmere (96), Inner Mongolia White Cashmere (40), and Chengdu Grey (42) breeds in China were genotyped at GHR locus using the protocol developed. In all goat breeds investigated, a SNP in exon 10 of GHR gene has been identified by analyzing genomic DNA. The polymorphism consists of a single nucleotide substitution A â†’ G, resulting in two alleles named, respectively, A and G based on the nucleotide at the position. The allele A was found to be more common in the animals investigated, and seems to be more consistent with cattle and zebu at this polymorphic site found in goats. The Hardy-Weinberg equilibrium of genotype distributions of GHR locus was verified in Liaoning Cashmere, and Inner Mongolia White Cashmere breeds. According to the classification of polymorphism information content (PIC), Chengdu Grey was less polymorphic than Liaoning Cashmere and Inner Mongolia White Cashmere breeds at this locus. The phylogenetic tree of different species based on the nucleotide sequences of GHR gene exon 10 is generally in agreement with the known species relationship. No significant association was found between the polymorphism revealed and the cashmere traits analyzed in present work.

Identification and expression analysis of two growth hormone receptors in zanzibar tilapia (Oreochromis hornorum).

Growth hormone plays important roles in various physiological processes such as growth, metabolism, and reproduction. In this study, two cDNAs encoding growth hormone receptor (GHR) were isolated from the liver of zanzibar tilapia (Oreochromis hornornum). The two cDNAs were 2,831 and 2,044 bp in length and named GHR1 and GHR2, respectively. GHR1 and GHR2 shared 57.4% similarity in nucleotide sequences and 33.5% similarity in deduced amino acid sequences. Consequently, it was presumed that they were two different genes. Conserved regions of GHR1 and GHR2 in zanzibar tilapia were different from those of other vertebrates. For example, conserved box2 regions of GHR1 and GHR2 in zanzibar tilapia were, respectively, WVELM and WVEFT, while it was WVEFI for GHRs in other vertebrates. Similar to other fish species, GHR1 and GHR2 were expressed in brain, gill, liver, muscle, spleen, gonad, stomach, kidney, and pituitary in zanzibar tilapia. The expression levels were the highest in liver. Unlike fathead minnow (Pimephales promelas) and mossambique tilapia (O. mossambicus), the expression levels of GHR1 in most female fish tissues were higher than those in male fish. No significant difference in GHR2 expression was found in all the tissues in male and female of zanzibar tilapia. Under fasting condition, the expressions of GHRs and IGF-II were significantly up-regulated (P < 0.05) in liver, while the expression of IGF-I remained stable. This observation would contribute to understanding the evolution of the GHR family in further investigation of growth regulation of zanzibar tilapia.

GHR signalling: Receptor activation and degradation mechanisms.

Growth hormone (GH) actions via initiating cell signalling through the GH receptor (GHR) are important for many physiological processes, in addition to its well-known role in regulating growth. The activation of JAK-STAT signalling by GH is well characterized, however knowledge on GH activation of SRC family kinases (SFKs) is still limited. In this review we summarise the collective knowledge on the activation, regulation, and downstream signalling of GHR. We highlight studies on GH activation of SFKs and the important outcome of this signalling pathway with a focus on the different degradation mechanisms that can regulate GHR availability since this is an area that warrants further study considering its role in tumour progression.

Turning the growth hormone receptor on: evidence that hormone binding induces subunit rotation.

Atomistic molecular dynamics simulations have been used to investigate the conformational changes associated with the binding of human growth hormone (hGH) to the extracellular domains (ECD) of the human growth hormone receptor (hGHR), thereby shedding light on the mechanism of activation. It is shown that the removal of hGH from the hormone-bound receptor complex results in a counter-clockwise rotation of the twosubunits relative to each other by 30 degrees -64 degrees (average 45 degrees +/- 14 degrees), in close agreement in terms of both the magnitude and direction of the rotation with that proposed based on mutagenesis experiments. In addition to providing evidence to support a rotational activation mechanism, the simulations have enabled the nature of the interaction interfaces in both the cytokine-bound and unliganded hGHR states to be analyzed in detail.

Purification of recombinant growth hormone by clear native gels for conformational analyses: preservation of conformation and receptor binding.

Most protein preparations require purification steps prior to biophysical analysis assessing protein stability, secondary structure and degree of folding. It was, therefore, the aim of this study to develop a system to separate and purify a protein from a commercially available medicinal product, recombinant human growth hormone (rhGH) and show preservation of conformation and function following the gel-based procedure. The rhGH was run on clear native (CN) gels and recovered from the gels by electroelution using D-Tube Dialyzer Midi under rigorous cooling. Melting point studies indicated preservation of the structural integrity. This finding was confirmed by synchrotron radiation circular dichroism spectroscopy (SRCD) revealing an identical folding pattern for the sample before and after electrophoretic separation and purification. Synchrotron small-angle X-ray scattering (SAXS) indicated that the sample was folded and monomeric, both before and after separation and purification, and that its shape corresponded well to the known crystal structure of GH. Binding properties of rhGH to a receptor-model system before and after clear native electrophoresis were comparable. This analytical and preparative approach to purify and concentrate a protein preserving conformation and function may be helpful for many applications in analytical, protein and stereochemistry.

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer.

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Prediction of protein-protein interface sequence diversity using flexible backbone computational protein design.

A major challenge in computational protein design is to identify functional sequences as top predictions. One reason for design failures is conformational plasticity, as proteins frequently change their conformation in response to mutations. To advance protein design, here we describe a method employing flexible backbone ensembles to predict sequences tolerated for a protein-protein interface. We show that the predictions are enriched in functional proteins when compared to a phage display screen quantitatively mapping the energy landscape for the interaction between human growth hormone and its receptor. Our model for structural plasticity is inspired by coupled side chain-backbone "backrub" motions observed in high-resolution protein crystal structures. Although the modeled structural changes are subtle, our results on predicting sequence plasticity suggest that backrub sampling may capture a sizable fraction of localized conformational changes that occur in proteins. The described method has implications for predicting sequence libraries to enable challenging protein engineering problems.

Yeast recombinant production of intact human membrane proteins with long intrinsically disordered intracellular regions for structural studies.

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations.

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.