Agrin

Agrin is a large is_associated_with::proteoglycan whose best-characterised role is in the development of the is_associated_with::neuromuscular junction during is_associated_with::embryogenesis. Agrin is named based on its involvement in the aggregation of is_associated_with::acetylcholine receptors during is_associated_with::synaptogenesis. In humans, this protein is encoded by the AGRN gene.

This protein has nine domains homologous to protease inhibitors. It may also have functions in other tissues and during other stages of development. It is a major is_associated_with::proteoglycan component in the glomerular basement membrane and may play a role in the renal filtration and cell-matrix interactions.

Discovery
Agrin was first identified by the U.J. McMahan laboratory, Stanford University.

Mechanism of action
During development, the growing end of motor neuron axons secrete a protein called agrin. This protein binds to several receptors on the surface of skeletal muscle. The receptor that seems to be required for formation of the is_associated_with::neuromuscular junction (NMJ) is called the is_associated_with::MuSK receptor (Muscle specific kinase). MuSK is a receptor tyrosine kinase - meaning that it induces cellular signaling by causing the addition of phosphate molecules to particular tyrosines on itself and on proteins that bind the cytoplasmic domain of the receptor.

In addition to MuSK, agrin binds several other proteins on the surface of muscle, including is_associated_with::dystroglycan and is_associated_with::laminin. It is seen that these additional binding steps are required to stabilize the NMJ.

The requirement for Agrin and MuSK in the formation of the NMJ was demonstrated primarily by is_associated_with::knockout mouse studies. In mice that are deficient for either protein, the neuromuscular junction does not form. Many other proteins also comprise the NMJ, and are required to maintain its integrity. For example, MuSK also binds a protein called "dishevelled" (Dvl), which is in the is_associated_with::Wnt signalling pathway. Dvl is additionally required for MuSK-mediated clustering of AChRs, since inhibition of Dvl blocks clustering.

Signaling
The nerve secretes agrin, resulting in phosphorylation of the is_associated_with::MuSK receptor.

It seems that the is_associated_with::MuSK receptor recruits is_associated_with::casein kinase 2, which is required for clustering.

A protein called is_associated_with::rapsyn is then recruited to the primary MuSK scaffold, to induce the additional clustering of acetylcholine receptors (AChR). This is thought of as the secondary scaffold. A protein called is_associated_with::Dok-7 has shown to be additionally required for the formation of the secondary scaffold; it is apparently recruited after MuSK phosphorylation and before acetylcholine receptors are clustered.

Structure
There are three potential is_associated_with::heparan sulfate (HS) attachment sites within the primary structure of agrin, but it is thought that only two of these actually carry HS chains when the protein is expressed.

In fact, one study concluded that at least two attachment sites are necessary by inducing synthetic agents. Since agrin fragments induce acetylcholine receptor aggregation as well as phosphorylation of the MuSK receptor, researchers spliced them and found that the variant did not trigger phosphorylation. It has also been shown that the G3 domain of agrin is very plastic, meaning it can discriminate between binding partners for a better fit.

Heparan sulfate glycosaminoglycans covalently linked to the agrin protein have been shown to play a role in the clustering of AChR. Interference in the correct formation of heparan sulfate through the addition of chlorate to skeletal muscle cell culture results in a decrease in the frequency of spontaneous acetylcholine receptor (AChR) clustering. It may be that rather than solely binding directly to the agrin protein core a number of components of the secondary scaffold may also interact with its heparan sulfate side-chains.

A role in the retention of anionic macromolecules within the vasculature has also been suggested for agrin-linked HS at the is_associated_with::glomerular or alveolar is_associated_with::basement membrane.