GHBP Rat
Description
GHBP is expressed with an 8 amino acid His tag at C-Terminus and purified by proprietary chromatographic techniques.
Product Specs
Growth hormone receptor (GHR) belongs to the cytokine receptor family. GHR binds two receptor molecules, leading to signal transduction via receptor dimerization. At elevated concentrations, growth hormone (GH) exhibits antagonist properties due to significant differences in binding affinities at the respective sites. This antagonistic effect can be further enhanced by reducing binding at the low-affinity site.
GHBP, GH receptor.
Q&A
What is GHBP in rats and how does it differ from other species?
GHBP in rats is a soluble protein found in serum that specifically interacts with growth hormone. Unlike humans and several other species where GHBP is derived from proteolytic processing of the growth hormone receptor (GHR), rats have a unique mechanism. In rats, GHBP contains the extracellular portion of the GHR but is produced from a distinct mRNA that contains an alternatively spliced exon. This exon replaces the transmembrane and intracellular domains of the receptor with a short hydrophilic carboxy-terminus of 17 amino acids .
This species difference is significant for comparative endocrinology research and highlights the importance of caution when extrapolating findings across species. Mice show a similar mechanism to rats but with a slightly longer C-terminal sequence of 25 amino acids .
What is the molecular structure and biochemical profile of rat GHBP?
Rat GHBP exists in two primary forms with apparent molecular weights of 52 and 44 kDa. Both forms arise from a single protein core of approximately 32 kDa through extensive glycosylation . This differential glycosylation may have functional significance that remains to be fully elucidated.
The protein concentration in rat serum shows significant sexual dimorphism:
| Sex | GHBP Concentration (ng/ml)* |
|---|---|
| Male | 300 |
| Female | 575 |
*Measured in nonglycosylated GHBP equivalents
Where is GHBP expressed in rat tissues?
GHBP in rats is predominantly expressed in:
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Serum - as freely circulating protein
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Growth plate chondrocytes - with specific distribution patterns:
Immunohistochemical studies reveal that GHBP is localized in both the cytoplasm and nucleus of these cells, suggesting potential intracellular functions beyond its role as a circulating protein .
What techniques are most effective for detecting GHBP in rat tissues?
Several sophisticated methodologies have been validated for studying rat GHBP:
Immunohistochemistry with Tyramide Signal Amplification (TSA)
This enhanced sensitivity technique utilizes antibodies specific for GHBP to visualize its presence in tissue sections. The TSA method significantly improves signal detection, enabling clear visualization of GHBP in different chondrocyte populations and subcellular compartments .
Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
RT-PCR provides confirmation of GHBP mRNA expression in tissues like the growth plate. This technique is essential for verifying that observed protein originates from specific GHBP transcripts rather than from GHR proteolysis .
Specialized Antibody Development
The generation of antibodies that can distinguish between GHBP and GHR is critical. Monoclonal antibodies such as GHBP 4.3 have been developed using synthetic peptides containing the unique C-terminal 17 amino acids of rat GHBP as immunogens . These antibodies are specific to rat GHBP and do not cross-react with rat GHR.
How can researchers generate specific antibodies to rat GHBP?
Generating antibodies that specifically recognize rat GHBP without cross-reacting with GHR presents a significant challenge due to the shared extracellular domain. Several innovative approaches have been developed:
Direct Peptide Immunization
This approach uses synthetic peptides containing the unique C-terminal sequence of rat GHBP (17 amino acids) as immunogens to raise monoclonal antibodies. This strategy targets the only region that differentiates GHBP from GHR .
Epitope-Switching Strategy
This sophisticated technique involves interspecies switching between rat and ovine growth hormone receptors:
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Identification of dominant linear epitopes in ovine GHBP
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Site-directed mutagenesis to alter these epitopes to the analogous sequences in rat GHBP
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Bacterial expression and purification of mutant proteins
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Immunization to generate antisera in sheep
This approach has successfully generated antibodies specific to rat GHBP, particularly when targeting site A (between Thr28 and Leu34) .
| Epitope Site | Location in Protein | Equivalent in Ovine GHBP |
|---|---|---|
| Site A | Between Thr28 and Leu34 | Epitope 1 |
| Site B | Between Ser121 and Asp124 | Epitope 5 |
Only the site A mutant protein elicited a specific anti-rat GHBP response in immunization studies .
How is rat GHBP expression regulated by hormones?
The regulation of GHBP in rats shows complex hormonal dependencies:
Growth Hormone (GH)
Studies using GH-deficient dwarf rats reveal reduced GHBP staining compared to normal rats, suggesting positive regulation by GH .
Thyroid Hormones (TH)
Administration of thyroid hormones (T3 + T4) to hypophysectomized (Hx) rats induces:
Combined GH and TH Treatment
When hypophysectomized rats receive both GH and thyroid hormones:
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Greater growth is observed
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A broader layer of GHBP-positive cells develops
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The pattern becomes indistinguishable from normal rats
This suggests synergistic effects of these hormones on GHBP expression .
Glucocorticoids
Dexamethasone treatment in normal rats:
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Inhibits growth
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Reduces GHBP expression
These findings indicate negative regulation by glucocorticoids .
How does GHBP expression change during development in rats?
Rat GHBP expression shows significant age-dependent patterns:
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Immunohistochemical staining for GHBP decreases with age
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In 12-week-old normal rats, only the early maturing chondrocytes demonstrate positive staining
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Younger rats exhibit more widespread staining across growth plate regions
These changes correlate with the natural slowing of growth as rats mature, suggesting functional relationships between GHBP expression and growth velocity.
What animal models are most informative for studying rat GHBP function?
Several rat models have proven valuable for GHBP research:
Normal Rats at Different Ages
These models help establish baseline expression patterns and age-related changes .
GH-Deficient Dwarf Rats
These rats show reduced GHBP staining, providing insights into GH-dependent regulation .
Hypophysectomized (Hx) Rats
With surgical removal of the pituitary gland, these rats allow investigation of multiple pituitary hormone effects on GHBP expression. In these rats, GHBP is clearly reduced, confirming pituitary hormone dependency .
Hormone Replacement Models
Administering specific hormones to Hx rats via subcutaneously implanted osmotic minipumps enables precise studies of individual hormone effects and interactions .
What factors can confound GHBP measurements in rat studies?
Several experimental variables can significantly impact GHBP measurements:
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Age of rats: Expression decreases with age, necessitating age-matched controls
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Sex differences: Female rats have approximately 1.9× higher serum levels than males
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Nutritional status: May affect GHBP expression but requires further investigation
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Tissue preparation methods: Critical for preserving epitope integrity
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Antibody specificity: Essential to distinguish GHBP from GHR
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Glycosylation detection: Different assays may detect various glycosylated forms with varying efficiency
Researchers should carefully control and report these variables to ensure reproducible results.
What is the relationship between GHBP and growth plate development?
GHBP exhibits a specific localization pattern in the rat growth plate, with strongest expression in early maturing chondrocytes at the interface between proliferative and hypertrophic zones . This strategic positioning suggests GHBP may play a role in:
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Modulating local GH availability to specific chondrocyte populations
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Potentially participating in the transition from proliferation to maturation
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Contributing to the "dual effector theory" of GH action, where GH acts primarily on "stem cells" of the growth plate
The nuclear localization of GHBP in growth plate chondrocytes also raises intriguing questions about potential direct genomic actions that remain to be fully characterized .
How might the unique production mechanism of rat GHBP impact experimental design?
The alternative splicing mechanism that generates rat GHBP from a unique mRNA (rather than from GHR proteolysis as in humans) has significant implications for research:
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Translational limitations: Findings about GHBP regulation in rats may not directly apply to humans
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Genetic manipulation approaches: Gene editing studies must account for the shared exons between GHR and GHBP
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Therapeutic development: Rat models for testing GHBP-targeted interventions may have limited human relevance
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Evolutionary considerations: The distinct mechanisms raise questions about the evolutionary advantages of different GHBP production strategies
Researchers must carefully consider these species differences when designing studies and interpreting results for potential human applications.
Product Science Overview
Growth Hormone Binding Protein (GHBP) is a crucial component in the regulation of growth hormone (GH) activity. GHBP is derived from the extracellular domain of the Growth Hormone Receptor (GHR) and exists in the bloodstream, where it binds to GH, modulating its availability and activity. The recombinant form of GHBP, specifically from rats, has been extensively studied to understand its role in GH physiology and its potential therapeutic applications.
GHBP is a soluble protein that binds to GH with high affinity, similar to the membrane-bound GHR. The binding of GH to GHBP can prolong the half-life of GH in the bloodstream, thereby enhancing its biological activity. This interaction is crucial for maintaining the appropriate levels of GH in the body and ensuring its proper physiological functions.
Recombinant GHBP can be produced using various expression systems, including bacterial, yeast, and mammalian cells. The most common method involves the use of Escherichia coli (E. coli) due to its simplicity and cost-effectiveness. The gene encoding GHBP is cloned into an expression vector, which is then introduced into E. coli cells. The bacteria are cultured, and the recombinant protein is expressed, harvested, and purified using techniques such as affinity chromatography.
Studies have shown that recombinant GHBP can enhance the growth-promoting activity of GH in rats. For instance, coadministration of recombinant human GHBP (rhGHBP) with recombinant human GH (rhGH) in hypophysectomized rats or GH-deficient dwarf rats resulted in increased body weight gain and bone growth . This effect is attributed to the ability of GHBP to slow the clearance of GH from the bloodstream, thereby increasing its bioactivity.
The localization and regulation of GHBP in the rat growth plate have been investigated using immunohistochemical methods. GHBP is found in various layers of the growth plate, including germinal and proliferative chondrocytes, as well as early maturing chondrocytes . The expression of GHBP is regulated by hormonal factors, such as thyroid hormones and glucocorticoids, which can influence its levels and activity in the growth plate.
The ability of GHBP to modulate GH activity has significant therapeutic implications. By enhancing the bioactivity of GH, recombinant GHBP could potentially be used to treat conditions associated with GH deficiency, such as dwarfism and growth retardation. Additionally, understanding the role of GHBP in GH physiology could lead to the development of novel therapeutic strategies for managing GH-related disorders.
