B7-33: RXFP1 Signalling, ERK Pathway Bias and Relaxin Peptide Structure

B7-33: RXFP1 Signalling, ERK Pathway Bias and Relaxin Peptide Structure

B7-33 is a synthetic single-chain peptide which has been derived from the B-chain of human relaxin-2 and is closely associated with the relaxin family peptide receptor 1, or RXFP1, a G protein-coupled receptor that is involved in relaxin signalling.

What is especially interesting about B7-33 from the point of view of molecular research is its signalling profile. Unlike native human relaxin-2, which activates a number of downstream pathways such as cyclic AMP and ERK signalling, B7-33 was developed as a functionally selective agonist for RXFP1. In experimental studies it has been found that ERK1/2 signalling is preferentially activated over cAMP in certain cellular systems.

B7-33 can therefore be used as a research tool when studying receptor signalling, pathway selectivity and extracellular matrix biology; although its published work also involves cardiovascular and fibrosis models, these studies must be regarded as preclinical experimental evidence and not as evidence of its efficacy in humans.

B7-33 and the Structure of Relaxin-2

Human relaxin-2 is a peptide hormone similar to insulin and has a structure composed of two chains linked together by disulfide bonds; the B-chain includes the residues that are involved in interaction with RXFP1.

The single-chain derivative of this B-chain region is B7-33. The molecule thus obtained is less complex in structure than native relaxin-2 but still exhibits activity at RXFP1.

The structural change is scientifically significant since the biological behaviour of a peptide is closely related to its sequence, three-dimensional structure, receptor-binding properties and conformational flexibility.

The study of B7-33 thus serves as an example of how a naturally occurring peptide hormone can be used as a starting point for the development of a smaller analogue which is active at the receptor.

It should always be kept in mind when interpreting the experimental results that B7-33 and native relaxin-2 are different molecules, since although they are related they are not structurally identical and do not necessarily lead to the same receptor signalling.

RXFP1 as the Primary Research Target

RXFP1 belongs to the group of G protein-coupled receptors and is responsible for many of the biological effects linked with relaxin-2.

The receptor has a complicated structure in the region outside the cell and is able to interact with various intracellular signalling systems according to the ligand, the cellular environment and the receptor context.

Native relaxin signalling is linked to the cAMP, cGMP and mitogen-activated protein kinase pathways, and studies have found interactions between RXFP1 and other receptors, such as the angiotensin II type 2 receptor in some cellular systems.

The B7-33 method allows one to examine whether specific activation of particular RXFP1 signalling branches leads to different molecular responses.

One of the reasons why B7-33 is of greater scientific interest is that it can be described merely as a shorter form of relaxin.

B7-33 and Functional Selectivity at RXFP1

The phenomenon known as functional selectivity refers to the case where various ligands, when acting on the same receptor, prefer to activate different downstream signalling pathways.

The study based on B7-33 looked into this concept by using cellular systems that expressed RXFP1; in these systems B7-33 caused phosphorylation of ERK1/2 but generated considerably less cAMP signalling than native H2 relaxin.

It is important to make this distinction since the activation of a receptor is not always a simple, linear process; a ligand can affect the conformation of the receptor and thus change the intracellular signalling pathways that are preferentially recruited.

ERK1/2 phosphorylation became an important experimental endpoint for B7-33 since it offered a measurable indicator of pathway-selective RXFP1 activity.

The extent of selectivity also varies with the experimental system; B7-33 exhibited different signalling properties in cells that had overexpressed RXFP1 as compared to those that naturally expressed the receptor. This demonstrates the importance of receptor density and cellular context when evaluating peptide activity.

ERK1/2 Signalling in B7-33 Research

ERK1/2 is a member of the mitogen-activated protein kinase family and is involved in the intracellular pathways that control processes such as gene expression, cellular differentiation and responses of the extracellular matrix.

In the B7-33 experiments phosphorylation of ERK1/2 has served as a marker of receptor-linked signalling.

In the initial studies it was found that there was a quick rise in the phosphorylation of ERK1/2 when the cells expressing RXFP1 were exposed to B7-33. Later experiments then linked this signalling pathway with matrix metalloproteinase activity and extracellular matrix remodelling in the experimental models.

The fact that these observations have is that ERK activation does not have a single universal biological effect; instead, ERK signalling is highly dependent on the context and may lead to different results depending on the cell type, the level of receptor expression, the duration of the signalling and its interactions with other pathways.

As a result, ERK1/2 phosphorylation ought to be regarded as a molecular endpoint rather than as an independent sign of a therapeutic effect.

RXFP1 and AT2R Receptor Crosstalk

A relatively specific aspect of B7-33 research is the study of the interaction between RXFP1 and the angiotensin II type 2 receptor, or AT2R.

Studies on relaxin signalling have shown there to be functional interactions between these receptor systems in fibroblast models. Investigations have been carried out into the relationship between B7-33 and RXFP1-AT2R signalling and the subsequent ERK1/2 activity.

It is for this reason that peptide signalling cannot always be understood by looking at a single receptor on its own.

The reaction at the cellular level could be due to receptor complexes, intracellular phosphatases, kinase activity and the presence of other signalling partners.

It therefore follows that when laboratory studies are carried out, measuring both RXFP1 activation and the downstream pathway markers will yield more information than measuring just a single endpoint.

B7-33 and Extracellular Matrix Research

The biology of the extracellular matrix is also a major area of interest in relation to B7-33 and the wider relaxin signalling system.

Fibroblasts carry out the production and modification of a number of the components of the extracellular matrix, such as collagen; when they become activated myofibroblasts there occur changes in matrix production and restructuring.

Research into relaxin signalling has been carried out with regard to collagen metabolism and matrix metalloproteinases, and similarly the B7-33 study has looked at matrix-associated endpoints in fibroblast and animal models.

Matrix metalloproteinase-2, or MMP-2, is of particular relevance since it is involved in the turnover of the extracellular matrix. In certain models, experimental studies have linked B7-33-induced RXFP1 signalling with MMP-2 activity.

It offers a molecular method by which one can examine how selective receptor signalling can affect extracellular matrix processes without having to assume that each downstream response is the same in different tissues.

B7-33 in Cardiac Experimental Models

While the main aim of this article in a commercial sense is 'heart protection', the scientific literature should actually be seen as a set of preclinical cardiac experiments.

B7-33 has been studied in a number of rodent models that involved myocardial injury, cardiomyopathy and cardiac fibrosis, and the experiments looked at myocardial collagen deposition, left ventricular structure, vascular density, inflammatory markers and cardiac functional measurements.

For example,

research published in 2019 investigated B7-33 in a mouse model of myocardial ischemia-reperfusion injury and reported changes in infarct size and cardiac functional measurements compared with vehicle controls.

A further study looked at B7-33 in an experimental model of cardiomyopathy and measured the level of fibrosis, the degree of cardiomyocyte hypertrophy, the vascular changes and the levels of inflammatory markers.

The fact that these findings exist shows that research into B7-33 in the field of cardiovascular biology will continue. At the same time, they do not prove that B7-33 should be used as a treatment for humans.

Even though animal models are able to simulate certain aspects of cardiovascular pathology under controlled experimental conditions, they cannot be relied upon to prove clinical efficacy or to evaluate how a peptide would perform in different human populations.

B7-33 and Cardiac Fibroblast Research

Cardiac fibroblasts also offer a useful model for the study of the relationship between RXFP1 signalling and extracellular matrix biology.

Experimental studies using human cardiac fibroblasts have looked at relaxin signalling when subjected to profibrotic stimuli such as transforming growth factor beta.

The researchers have measured a number of endpoints such as myofibroblast differentiation, collagen production, receptor expression and intracellular signalling.

Research into relaxin itself has also looked at the interactions between RXFP1, AT2R and other signalling components. This kind of research helps to explain why B7-33 is being studied as a functionally selective relaxin analogue.

It is still important to distinguish between native relaxin and B7-33; whenever relaxin has been studied, the results obtained should not be automatically applied to B7-33 unless the B7-33 molecule has been tested directly.

Experimental Models Used in B7-33 Research

The research covered by B7-33 involves a number of experimental systems.

Studies based on cell lines that express RXFP1 have been carried out to examine receptor activation, the accumulation of cAMP and the phosphorylation of ERK1/2. In other experiments primary fibroblasts or cells which naturally express RXFP1 have been used.

Studies involving animals have looked at myocardial injury, cardiomyopathy, fibrosis and vascular responses. Since these models enable researchers to measure tissue-level endpoints which cannot be obtained from isolated cells.

Different models are suited to different questions; a receptor-expression system can be used for characterising signalling bias, while a primary fibroblast model might give more information regarding endogenous receptor behaviour. An animal model can then look at how various tissues interact within a complete biological system.

Comparisons involving these models should therefore take into account receptor expression, ligand concentration, exposure time, species, and the selection of endpoint.

B7-33 Research and Receptor Context

A major limitation when interpreting the B7-33 experiments is the receptor context.

A ligand can behave differently when a receptor is overexpressed compared with when the receptor occurs at its endogenous level. Receptor density can alter apparent potency and signalling responses, while receptor interactions can introduce additional pathways.

The study involving the B7-33 strain showed this difference by looking at the signalling in cells that had different characteristics regarding RXFP1 expression.

What this shows is that a single concentration-response curve fails to give a full account of peptide pharmacology since the number of receptors, the type of cell, the downstream signalling apparatus and the time of the experiment all have an effect on the response observed.

Analytical and Structural Considerations

The research involving B7-33 needs the peptide in question to be identified accurately.

It is important to consider sequence identity and to carry out molecular characterisation when comparing research materials or experiments that have been published, since B7-33 is derived from the B-chain of human relaxin-2.

Analytical techniques such as high-performance liquid chromatography and mass spectrometry are capable of being used to evaluate purity and molecular identity; batch-specific analytical records can also give further information about the material used in a laboratory study.

The structural differences between B7-33, native relaxin-2 and the other relaxin-derived analogues should also be taken into account. The use of similar terminology does not mean that they are chemically equivalent.

Before making any comparison of biological observations, it is necessary to determine the exact peptide sequence, any modifications, the purity and the analytical profile.

Limitations of Current B7-33 Research

B7-33 has got a considerable background of preclinical research but some important limitations still exist.

A great deal of the evidence is obtained from studies using cells and animals. Although such studies are helpful for investigating receptor pharmacology, signalling pathways and tissue-level responses, they fail to demonstrate clinical efficacy.

The pharmacology of RXFP1 is likewise complicated. The signalling profiles that result can vary depending on the receptor environment, and receptor interactions may have an effect on the subsequent responses.

Another limitation is that evidence about native relaxin cannot be directly applied to B7-33. Since B7-33 was specifically designed as a structurally modified, functionally selective analogue of relaxin, direct experimental evidence is necessary if anything is to be claimed regarding its activity.

Research in the future may therefore still look at receptor structure, signalling bias, pharmacokinetics, peptide stability and tissue-specific responses.

Research Use Only

The B7-33 materials referred to in this article are for use solely in scientific and laboratory research, and are not meant for use by humans or animals in consumption, nor are they provided for the diagnosis, treatment, curing or prevention of any disease or medical condition.

The article does not include any information regarding dosage, instructions for administration or protocols suitable for use in humans; the studies on cardiovascular and fibrosis that are published aim to explain experimental research and molecular mechanisms and are not intended to promote therapeutic use.

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