GHBP Rabbit

Introduction to GHBP in Rabbits

Growth Hormone Binding Protein (GHBP) is a soluble protein corresponding to the extracellular domain of the growth hormone receptor (GHR). In rabbits, GHBP serves as a transmembrane receptor for growth hormone, with binding leading to receptor dimerization and activation of intra- and intercellular signal transduction pathways that promote growth . The rabbit GHBP system has been extensively studied as a model for understanding growth hormone regulation across species, particularly due to its similarities with human GHBP mechanisms. Unlike rodent models, rabbit GHBP generation occurs through proteolytic mechanisms rather than alternative splicing, making it an important comparative model for human growth hormone studies . This protein plays vital roles in modulating circulating growth hormone levels, affecting its bioavailability, and influencing growth and metabolic processes throughout the body.

Proteolytic Generation

Unlike in rodents where GHBP is generated through alternative mRNA splicing, rabbits produce GHBP through proteolytic cleavage of the membrane-bound growth hormone receptor (GHR) . This mechanism represents a critical distinction in species-specific GHBP generation, sharing similarities with the human system rather than that of mice and rats . The proteolytic process results in the shedding of the extracellular domain of the GHR, yielding a soluble GHBP that maintains growth hormone binding capacity.

Role of Metalloproteases

Research has identified TACE/ADAM-17 (tumor necrosis factor-alpha-converting enzyme) as a critical metalloprotease involved in GHBP shedding in rabbits . Studies using Chinese hamster ovary cells transfected with rabbit GHR complementary DNA have demonstrated that PMA (phorbol 12-myristate,13-acetate) induces GHBP shedding through TACE-mediated mechanisms . Experiments with TACE knockout models revealed that TACE reconstitution was necessary for PMA-induced GHBP generation, establishing this enzyme as an essential component in the proteolytic release of rabbit GHBP .

Energy and Temperature Dependence

The release of GHBP from rabbit GHR is an energy-requiring process with significant temperature dependence. Research has shown that GHBP release is reduced at low incubation temperatures, suggesting that the cleavage mechanism requires active cellular processes . When observed in experimental settings, GHBP secretion becomes detectable in the medium at approximately 30 minutes, with a linear increase over the following 4 hours, demonstrating the time-dependent nature of this process .

Functional Roles of Rabbit GHBP

Rabbit GHBP serves multiple physiological functions that collectively impact growth hormone signaling and activity:

Hormone Binding and Transport

GHBP binds approximately half of the circulating growth hormone under basal conditions, though it becomes easily saturated at high growth hormone levels . This binding creates a circulating reservoir of the hormone, affecting its distribution, availability, and half-life in the bloodstream.

Modulation of Growth Hormone Activity

The binding of GHBP to growth hormone creates complex functional effects that include:

• Acting as a circulating buffer/reservoir for growth hormone

• Prolonging plasma growth hormone half-life

• Competing with growth hormone receptors for available growth hormone

• Potentially forming unproductive heterodimers with GHR

These diverse and sometimes opposing functions result in a complex net effect on growth hormone action in vivo that depends on physiological context and relative concentrations of the involved components.

Growth Regulation

Genetic studies investigating growth traits in different rabbit strains have demonstrated associations between growth hormone receptor gene polymorphisms and growth parameters. Research findings suggest that specific GHR genotypes correlate with differences in body weight and other growth metrics across various rabbit strains (New Zealand White, Dutch, and Hylamax) . These observations indirectly highlight the importance of the GHR-GHBP system in regulating growth processes in rabbits.

Factors Affecting GHBP Production

The secretion of GHBP from cells expressing rabbit GHR is influenced by multiple factors, including:

• Time of incubation (progressive increase over several hours)

• Percentage of fetal calf serum in culture media

• Temperature (reduced at lower temperatures)

• Protein synthesis capacity

Chemical Modulators of GHBP Release

Several chemical compounds affect the release of GHBP from rabbit GHR:

The effects of these compounds provide insights into the regulatory mechanisms controlling GHBP generation. For example, treatment with cycloheximide causes parallel disappearance of cellular GHR and secreted GHBP but increases GHR messenger RNA expression (superinduction) . Following removal of cycloheximide, GHR and GHBP levels increased by 181% and 369%, respectively, compared to control values, demonstrating complex regulatory dynamics .

Recombinant Rabbit GHBP

Recombinant rabbit GHBP is commercially available as a research tool, typically produced in Escherichia coli expression systems. These products contain the extracellular domain of rabbit growth hormone receptor and are purified through chromatographic techniques . Commercial preparations of rabbit GHBP are available in various quantities and concentrations for research purposes, with pricing typically ranging from approximately 100€ for 5 μg to over 8,000€ for 1 mg .

Formulation and Stability

Commercial rabbit GHBP is typically lyophilized from a concentrated solution (1mg/ml) with 0.0045mM NaHCO₃ and is recommended to be reconstituted in sterile 0.4% NaHCO₃ at pH 10, at concentrations not less than 100μg/ml . For optimal stability, reconstituted GHBP should be stored at 4°C for short-term use (2-7 days) and below -18°C for longer storage. Addition of carrier proteins such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) is recommended for long-term storage, and freeze-thaw cycles should be avoided .

Research Applications

Rabbit GHBP has been extensively used in research settings to:

• Study mechanisms of GHBP generation

• Explore growth hormone signaling pathways

• Develop antibodies against specific epitopes

• Create cellular models for investigating receptor-ligand interactions

• Compare species-specific differences in growth hormone regulation

Chinese hamster ovary cells expressing rabbit GHR have been established as a useful cellular model system for studying the mechanism of GHBP generation from GHR and its physiological importance . Such models provide valuable insights into the fundamental processes governing growth hormone signaling.

Comparative Analysis Across Species

Interestingly, while the primary mechanism of GHBP generation in rabbits involves proteolytic cleavage, research has also identified alternatively spliced forms in the cytoplasmic domain of GHR. One such form, designated GHR-tr, has been found in rabbits, mice, and rats . This transcript is identical in sequence to full-length GHR except for a 26-bp deletion leading to a stop codon, resulting in truncation of 97.5% of the intracellular domain . Studies suggest that this truncated form may have an increased capacity to generate soluble GHBP, indicating that multiple mechanisms may contribute to GHBP production across species.

Detection and Quantification Methods

Multiple approaches have been developed to study rabbit GHBP:

• Radioimmunological assays using polyclonal antibodies

• PCR-based detection of GHR variants

• Western blotting for protein identification

• Chromatographic separation techniques

• Cell-based assays using transfected cell lines

These methodological approaches have allowed researchers to characterize the properties, regulation, and function of rabbit GHBP in various experimental contexts. For instance, studies have employed Chinese hamster ovary cells transfected with rabbit GHR complementary DNA to study spontaneous release of GHBP into culture medium in relation to cellular GHR levels .

Genetic and Molecular Studies

Recent genetic studies have explored polymorphisms in the GHR gene across different rabbit strains, associating specific genotypes with growth traits. By targeting the GANTC restriction site, which is present twice in one allele (C) and once in another allele (G), researchers have identified genotypic frequencies of different rabbit strains and correlated these with growth parameters . These studies provide insights into how genetic variations in the GHR gene influence growth and potentially affect GHBP levels and function.