Aminosaurs, Complement, Embryology, Heparin, Neuroscience, Serine/Threonine, Tyrosine, Zinc
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Agrin is a large chimeric proteoglycan, a heparan sulfate and chondroitin proteoglycan, whose best-characterised role is in the development of the neuromuscular junction during embryogenesis

Agrin was originally found in the electric organ of Tarpedo california and in the basal lamina at the neuromuscular junction as a protein that directs the aggregation of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) at synaptic sites.

  • Agrin at ScienceDirect https://www.sciencedirect.com/topics/medicine-and-dentistry/agrin
Pacific electric ray (Torpedo californica), Kathy Dewet-Oleson, NOAA National Marine Sanctuaries

Tetronarce californica also known as the Pacific electric ray is a species of electric ray in the family Torpedinidae, endemic to the coastal waters of the northeastern Pacific Ocean from Baja California to British Columbia. It generally inhabits sandy flats, rocky reefs, and kelp forests from the surface to a depth of 200 m (660 ft), but has also been known to make forays into the open ocean. Measuring up to 1.4 m (4.6 ft) long, this species has smooth-rimmed spiracles (paired respiratory openings behind the eyes) and a dark gray, slate, or brown dorsal coloration, sometimes with dark spots. Its body form is typical of the genus, with a rounded pectoral fin disc wider than long and a thick tail bearing two dorsal fins of unequal size and a well-developed caudal fin. Solitary and nocturnal, the Pacific electric ray can generate up to 45 volts of electricity for the purposes of subduing prey or self-defense. It feeds mainly on bony fishes, ambushing them from the substrate during the day and actively hunting for them at night. Reproduction is aplacental viviparous, meaning that the embryos are initially nourished by yolk, later supplemented by histotroph (“uterine milk”) produced by the mother. Care should be exercised around the Pacific electric ray, as it has been known to act aggressively if provoked and its electric shock can potentially incapacitate a diver. It and other electric rays are used as model organisms for biomedical research because their electric organs contain an abundance of important nervous system proteins such as nicotinic acetylcholine receptor and acetylcholinesterase. In the 1970s and 1980s, acetylcholine receptors from this species and the marbled electric ray (T. marmorata) became the first neurotransmitter receptors to be isolated and sequenced, in what is considered to be a landmark success in the field of neurobiology. This led to a number of further advances, one of the most significant being the elucidation of the pathophysiology underlying the disease myasthenia gravis. 

  • Histotrophy is a form of matrotrophy exhibited by some live-bearing sharks and rays, in which the developing embryo receives additional nutrition from its mother in the form of uterine secretions, known as histotroph (or “uterine milk”). It is one of the three major modes of elasmobranch reproduction encompassed by “aplacental viviparity“, and can be contrasted with yolk-sac viviparity (in which the embryo is solely sustained by yolk) and oophagy (in which the embryo feeds on ova). There are two categories of histotrophy:
    • In mucoid or limited histotrophy, the developing embryo ingests uterine mucus or histotroph as a supplement to the energy supplies provided by its yolk sac. This form of histotrophy is known to occur in the dogfish sharks (Squaliformes) and the electric rays (Torpediniformes), and may be more widespread.
    • In lipid histotrophy, the developing embryo is supplied with protein and lipid-enriched histotroph through specialized finger-like structures known as trophonemata. The additional nutrition provided by the enriched histotroph allows the embryo to increase in mass from the egg by several orders of magnitude by the time it is born, much greater than is possible in mucoid histotrophy. This form of histotrophy is found in stingrays and their relatives (Myliobatiformes).
Fetal-maternal interface showing uterine milk

Uterine glands or endometrial glands are tubular glands, lined by a simple columnar epithelium, found in the functional layer of the endometrium that lines the uterus. Their appearance varies during the menstrual cycle. During the proliferative phase, uterine glands appear long due to estrogen secretion by the ovaries. During the secretory phase, the uterine glands become very coiled with wide lumens and produce a glycogen-rich secretion known as histotroph or uterine milk. This change corresponds with an increase in blood flow to spiral arteries due to increased progesterone secretion from the corpus luteum. During the pre-menstrual phase, progesterone secretion decreases as the corpus luteum degenerates, which results in decreased blood flow to the spiral arteries. The functional layer of the uterus containing the glands becomes necrotic, and eventually sloughs off during the menstrual phase of the cycle. They are of small size in the unimpregnated uterus, but shortly after impregnation become enlarged and elongated, presenting a contorted or waved appearance. Hormones produced in early pregnancy stimulate the uterine glands to secrete a number of substances to give nutrition and protection to the embryo and fetus, and the fetal membranes. These secretions are known as histiotroph, alternatively histotroph, and also as uterine milk. Important uterine milk proteins are glycodelin-A, and osteopontin. Some secretory components from the uterine glands are taken up by the secondary yolk sac lining the exocoelomic cavity during pregnancy, and may thereby assist in providing fetal nutrition.

The aggregating factor was named ‘agrin’ from the Greek word ‘ageirein’, which means ‘to assemble.’ In humans, this protein is encoded by the AGRN gene. Agrin is named based on its involvement in the aggregation of acetylcholine receptors during synaptogenesis.

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 proteoglycan component in the glomerular basement membrane and may play a role in the renal filtration and cell-matrix interactions.

Agrin functions by activating the MuSK protein (for Muscle-Specific Kinase),  which is a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Agrin is required to activate MuSK. Agrin is also required for neuromuscular junction formation.

MuSK (for Muscle-Specific Kinase) is a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. It is activated by a nerve-derived proteoglycan called agrin, which is similarly also required for neuromuscular junction formation.

Upon activation by its ligand agrin, MuSK signals via the proteins called casein kinase 2 (CK2), Dok-7 and rapsyn, to induce “clustering” of acetylcholine receptors (AChR). Both CK2 and Dok-7 are required for MuSK-induced formation of the neuromuscular junction, since mice lacking Dok-7 failed to form AChR clusters or neuromuscular synapses, and since downregulation of CK2 also impedes recruitment of AChR to the primary MuSK scaffold. In addition to the proteins mentioned, other proteins are then gathered, to form the endplate to the neuromuscular junction. The nerve terminates onto the endplate, forming the neuromuscular junction – a structure required to transmit nerve impulses to the muscle, and thus initiating muscle contraction.

Antibodies directed against this protein (Anti-MuSK autoantibodies) are found in some people with myasthenia gravis not demonstrating antibodies to the acetylcholine receptor. The disease still causes loss of acetylcholine receptor activity, but the symptoms affected people experience may differ from those of people with other causes of myasthenia gravis.[citation needed]

  • Hoch W, McConville J, Helms S, Newsom-Davis J, Melms A, Vincent A (Mar 2001). “Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies”. Nature Medicine. 7 (3): 365–8. doi:10.1038/85520. PMID 11231638. S2CID 18641849.
  • Barrett-Jolley R, Byrne N, Vincent A, Newsom-Davis J (Oct 1994). “Plasma from patients with seronegative myasthenia gravis inhibit nAChR responses in the TE671/RD cell line”. Pflügers Archiv. 428 (5–6): 492–8. doi:10.1007/BF00374570. PMID 7838671. S2CID 5611563.
Strabismus in a person with myasthenia gravis trying to open their eyes.

Myasthenia gravis (MG) is a long-term neuromuscular junction disease that leads to varying degrees of skeletal muscle weakness. The most commonly affected muscles are those of the eyes, face, and swallowing. It can result in double vision, drooping eyelids, and difficulties in talking and walking. Onset can be sudden. Those affected often have a large thymus or develop a thymoma. Myasthenia gravis is an autoimmune disease of the neuro-muscular junction which results from antibodies that block or destroy nicotinic acetylcholine receptors (AChR) at the junction between the nerve and muscle. This prevents nerve impulses from triggering muscle contractions. Most cases are due to immunoglobulin G1 (IgG1) and IgG3 antibodies that attack AChR in the postsynaptic membrane, causing complement-mediated damage and muscle weakness. Rarely, an inherited genetic defect in the neuromuscular junction results in a similar condition known as congenital myasthenia.

  1. Myasthenia Gravis Fact Sheet”. National Institute of Neurological Disorders and Stroke.
  2. Kahan S (2005). In a Page: Neurology. Lippincott Williams & Wilkins. p. 118. ISBN 978-1-4051-0432-6. Archived from the original on 8 September 2017.
  3. Kaminski HJ (2009). Myasthenia Gravis and Related Disorders (2 ed.). Springer Science & Business Media. p. 72. ISBN 978-1-59745-156-7. Archived from the original on 8 September 2017.
  4. Adams JG (2012). Emergency Medicine: Clinical Essentials (2 ed.). Elsevier Health Sciences. p. 844. ISBN 978-1-4557-3394-1. Archived from the original on 8 September 2017.
  5. Salari N, Fatahi B, Bartina Y, Kazeminia M, Fatahian R, Mohammadi P, et al. (December 2021). “Global prevalence of myasthenia gravis and the effectiveness of common drugs in its treatment: a systematic review and meta-analysis”. Journal of Translational Medicine. 19 (1): 516. doi:10.1186/s12967-021-03185-7. PMC 8686543. PMID 34930325.
  6. Young C, McGill SC (April 2021). Rituximab for the Treatment of Myasthenia Gravis: A 2021 Update [Internet] (Report). Ottawa (ON): Canadian Agency for Drugs and Technologies in Health. PMID 34255447.
  7. Dabi A, Solieman N, Kurukumbi M, Kalyanam J (2012). “Myasthenia Gravis: A Review”. Autoimmune Diseases. 2012: 1–10. doi:10.1155/2012/874680. PMC 3501798. PMID 23193443.
  8. Phillips WD, Vincent A (1 January 2016). “Pathogenesis of myasthenia gravis: update on disease types, models, and mechanisms”. F1000Research. 5: 1513. doi:10.12688/f1000research.8206.1. PMC 4926737. PMID 27408701.
  9. Kandel E, Schwartz J, Jessel T, Siegelbaum S, Hudspeth A (2012). Principles of Neural Science (5 ed.). pp. 318–319.
  10. Vrinten C, van der Zwaag AM, Weinreich SS, Scholten RJ, Verschuuren JJ (December 2014). “Ephedrine for myasthenia gravis, neonatal myasthenia and the congenital myasthenic syndromes”. The Cochrane Database of Systematic Reviews. 2014 (12): CD010028. doi:10.1002/14651858.CD010028.pub2. PMC 7387729. PMID 25515947.
ClassDescription
IAny eye muscle weakness, possible ptosis,
no other evidence of muscle weakness elsewhere
IIEye muscle weakness of any severity,
mild weakness of other muscles
IIaPredominantly limb or axial muscles
IIbPredominantly bulbar and/or respiratory muscles
IIIEye muscle weakness of any severity,
moderate weakness of other muscles
IIIaPredominantly limb or axial muscles
IIIbPredominantly bulbar and/or respiratory muscles
IVEye muscle weakness of any severity,
severe weakness of other muscles
IVaPredominantly limb or axial muscles
IVbPredominantly bulbar and/or respiratory muscles
VIntubation needed to maintain airway

Discovery

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

Mechanism of action

During development in humans, the growing end of motor neuron axons secrete a protein called agrin. When secreted, agrin binds to several receptors on the surface of skeletal muscle. The receptor which appears to be required for the formation of the neuromuscular junction (NMJ) is called the 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 dystroglycan and 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 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 Wnt signalling pathway. Dvl is additionally required for MuSK-mediated clustering of AChRs, since inhibition of Dvl blocks clustering.

Dishevelled (Dsh) is a family of proteins involved in canonical and non-canonical Wnt signalling pathways. Dsh (Dvl in mammals) is a cytoplasmic phosphoprotein that acts directly downstream of frizzled receptors. It takes its name from its initial discovery in flies, where a mutation in the dishevelled gene was observed to cause improper orientation of body and wing hairs. There are vertebrate homologs in zebrafish, Xenopus (Xdsh), mice (Dvl1, -2, -3) and humans (DVL-1, -2, -3). Dsh relays complex Wnt signals in tissues and cells, in normal and abnormal contexts. It is thought to interact with the SPATS1 protein when regulating the Wnt Signalling pathway.

Spermatogenesis associated serine rich 1 (SPATS1) is a protein which in humans is encoded by the SPATS1 gene. It is also known by the aliases Dishevelled-DEP domain interacting protein (DDIP), Spermatogenesis Associated 8 (SPATA8), and serin-rich spermatogenic protein 1 (SRSP1). A general idea of its chemical structure, subcellular localization, expression, and conservation is known. Research suggests SPATS1 may play a role in the canonical Wnt Signaling pathway and in the first spermatogenic wave. The expression of this protein has been found to greatly decline in adulthood, compared to expression levels measured in fetuses. Studies have shown some fluctuation during the gestation period, but overall remaining relatively high. There has also been evidence of high expression levels up until day 28 postpartum. Expression of this protein has been found in peritubular myoid cells, gonocytes, pachytene spermatocytes, spermatogonia, myoid cells, and Sertoli cells.

The image on the left represents a heat map of the expression levels of the SPATS1 protein in the pituitary gland. The picture on the right shows a scale for the color and shown and the coordinating expression level. These images were generated using Brain Allen.

Mouse brains have shown expression in various areas of the brain including the pituitary gland, the prefrontal cortex, the frontal lobe, the cerebellum, and the parietal lobe. Highest expression levels have been found in the testes, the next highest levels being found in the trachea. A protein abundance histogram, which compares the abundance of a desired protein to other proteins, shows that SPATS1 is on the lower level of expression.

Possible interacting proteins are listed in the table below. Note that these proteins have not been experimentally confirmed to interact with SPATS1. Instead, their interaction potential was determined by looking at concurrence patterns and textmining.

Protein NameFunctionScore
zinc finger protein 683
aka
ZNF683		may be involved in transcriptional regulation0.633
transmembrane channel like 5
aka TMC5		probable ion channel0.624
gametocyte specific factor 1 like
aka GTSF1L		unknown0.567
transmembrane protein 225
aka TMEM225		most likely inhibits phosphate 1 (PP1) in sperm
via binding to catalytic sub-unit PPP1CC		0.566
spermatogenesis associated 3
aka SPATA3		unknown0.537
family with sequence similarity71 member F1
aka FAM71F1		unknown0.535
chromosome 9 open reading frame 139
aka C9orf139		unknown0.477
sperm acrosome associated 4
aka SPACA4		sperm surface membrane protein that may be
involved in sperm – egg plasma
membrane adhesion and fusion during fertilization		0.472
sex comb on midleg-like protein 4
aka SCML4		PcG proteins that act by forming multi-protein
complexes, which are required to maintain the transcriptionally repressive state of homeotic genes throughout development		0.457
 “STRING Protein – Protein Interaction Tool”.

Dishevelled plays important roles in both the embryo and the adult, ranging from cellular differentiation and cell polarity to social behavior.

Members

There are three human genes that encode for the dishevelled proteins:

Overview of signal transduction pathways involved in apoptosis.

Function

DVL is an integral part of the Wnt canonical pathway (β-catenin dependent) and non-canonical pathway (β-catenin-independent). In either of these, DVL acts downstream of a Frizzled receptor, although the pathways are distinct.

Wnt canonical pathway

The Wnt canonical pathway, also known as the Wnt/β-catenin pathway, is activated during development, regulation, cell differentiation, and proliferation. The Wnt canonical pathway moves DVL between the cytoplasm and nucleus, via a conserved nuclear export sequence (NES) and a nuclear localization sequence (NLS), both necessary for proper functioning. The binding of Wnt to Frizzled receptors helps recruit DVL to the membrane, providing a site for Axin and GSK3β to bind and phosphorylate LRP5/6 (transmembrane low-density lipoprotein receptor-related protein), preventing constitutive degradation of β-catenin. The prevention of this degradation DVL allows for β-catenin buildup in the nucleus, where it acts as a coactivator for TCF (T cell factor) to activate Wnt responsive genes. Conversely, without Wnt signaling, the destruction complex, made of APC, CKI, GSK3β and Axin, degrades β-catenin buildup, keeping the concentration of β-catenin in the cell low.

Wnt non-canonical pathways

Planar cell polarity pathway

The planar cell polarity pathway (PCP) is the most notable β-catenin independent pathway – the Wnt signal is received by the Frizzled receptor, which relays signals to DVL, which then acts as a branch point for two independent pathways, leading to the activation of small GTPases Rho and Rac. For the Rho branch, Wnt signals induce DVL to form a complex with Daam1 (Dishevelled associated activator of morphogenesis 1). This complex then interacts with Rho guanine nucleotide exchange factor WGEF (weak-similarity GEF), which activates downstream effectors like Rho GTPase and Rho-associated kinase (ROCK), which activates actin and cytoskeleton architecture in the cell. For the Rac branch, DVL activates the Rac GTPase. Activating the Rac GTPase stimulates the downstream effector c-Jun N-terminal kinase (JNK), which controls rearrangements in the cytoskeleton and gene expression. More specifically, it regulates the polarity and movement of a cell, in processes in vertebrates (like Xenopus) including gastrulation, neural tube closure, and stereocilia orientation in the inner ear.

Wnt-calcium pathway

Another pathway independent of β-catenin is the Wnt-Ca2+ pathway, which is involved in cancer, inflammation, and neurodegeneration. Wnt triggers Frizzled-mediated activation, triggering a cascade leading to Ca2+ release, which activates effectors (e.g. CaMKII) that control gene transcription relevant to cell fate and cell migration. This pathway can switch off the Wnt/β-catenin cascade and it can also be inhibited by DVL activation.

There are five main highly conserved regions that exist in all variations of DVL. These include an amino-terminal DIX (N-terminus) domain, a PDZ (central) domain, a carboxyl-terminal DEP (C-terminus) domain, and two regions with positively charged amino acid residues. There is a proline-heavy region between the DIX and PDZ domains, and a largely basic region between the DIX and PDZ domains that has conserved serine and threonine residues. These regions mediate protein-protein interactions and help DVL channel signals into either the β-catenin or the β-catenin independent pathways. Additionally, there is the conserved nuclear export sequence (NES) and a nuclear localization sequence (NLS), whose ability to move DVL between the cytoplasm and the nucleus may be an important part of its function.

General structure of basic Dvl protein.

DIX domain (Dishevelled-Axin)

Located near the N-terminus region of DVL and consisting of about 82-85 amino acids for human DVL protein, DIX is found in proteins like Axin and coiled-coil protein DIX-domain-containing I (DIXdc1 or Ccd1). The DIX domain of DVL has five β-strands and one α helix with highly conserved amino acid residues.

PDZ domain

PDZ, whose name consists of the initials of first three identified proteins to share this common structural domain (Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and Zonula occludens-1 protein (zo-1)), lies in the central region of DVL. PDZ typically has about 73 amino acids in each human DVL protein, and consists of 5-6 β-strands and 2-3 α-helices  This motif plays a critical role in ligand binding and conformational properties of the DVL protein. This region mediates many protein-protein interactions and regulates multiple biological processes.

DEP domain (Dishevelled-EGL-10-Pleackstrin)

DEP, which is in C-terminal domain of DVL, has 75 amino acids in the human DVL proteins, and has three α-helices, a β-hairpin arm, and two short β-strands. This domain enables interaction between DVL and DAAM1, thus activating the non-canonical pathway. This domain also has results that support claims that the DEP domain is what is responsible for targeting DVL proteins to the membrane upon Wnt signal stimulation. The DEP domain may also be essential for the assembly of functional signalosomes and for Wnt signal transduction to the nucleus.

NES and NLS regions

In addition to these conserved regions, DVL has both a NES and NLS, which regulate the cellular localization of DVL via movement between the nucleus and the cytoplasm. The NLS is between the PDZ and DEP domains, and the NES is between the DEP and C-terminus of DVL.

There are three main types of DVL post-translational modification – phosphorylation, ubiquitination, and methylation. Phosphorylation is the most well-studied, and seems to act such that site-specific phosphorylation can bring about a wide variety of biological responses. Ubiquitination is the post-translational modification that has a role in regulating DVL degradation.

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

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

A protein called 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 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.

43 kDa receptor-associated protein of the synapse (rapsyn) is a protein that in humans is encoded by the RAPSN gene. This protein belongs to a family of proteins that are receptor associated proteins of the synapse. It contains a conserved cAMP-dependent protein kinase phosphorylation site. It is believed to play some role in anchoring or stabilizing the nicotinic acetylcholine receptor at synaptic sites. It may link the receptor to the underlying postsynaptic cytoskeleton, possibly by direct association with actin or spectrin. Two splice variants have been identified for this gene. In the neuromuscular junction there is a vital pathway that maintains synaptic structure and results in the aggregation and localization of the acetylcholine receptor (AChR) on the postsynaptic folds. This pathway consists of agrin, muscle-specific tyrosine kinase (MuSK protein), AChRs and the AChR-clustering protein rapsyn, encoded by RAPSN. Genetic mutations of the proteins in the neuromuscular junction are associated with Congenital myasthenic syndrome (CMS). Postsynaptic defects are the most frequent cause of CMS and often result in abnormalities in the acetylcholine receptor. The vast majority of mutations causing CMS are found in the AChR subunits and rapsyn genes.

The rapsyn protein interacts directly with the AChRs and plays a vital role in agrin-induced clustering of the AChR. Without rapsyn, functional synapses cannot be created as the folds do not form properly. Patients with CMS-related mutations of the rapsyn protein typically are either homozygous for N88K or heterozygous for N88K and a second mutation. The major effect of the mutation N88K in rapsyn is to reduce the stability of AChR clusters. The second mutation can be a determining factor in the severity of the disease.

Studies have shown that most patients with CMS that have rapsyn mutations carry the common mutation N88K on at least one allele. However, research has revealed that there is a small population of patients who do not carry the N88K mutation on either of their alleles, but instead have different mutations of the RAPSN gene on both of their alleles. Two novel missense mutations that have been found are R164C and L283P and the result is a decrease in co-clustering of AChR with raspyn. A third mutation is the intronic base alteration IVS1-15C>A and it causes abnormal splicing of RAPSN RNA. These results show that diagnostic screening for CMS mutations of the RAPSN gene cannot be based exclusively on the detection of N88K mutations Interestingly, patients who bear the burden of CMS due to these rapsyn mutations often demonstrate a remarkable response to anticholinesterase drugs like pyridostigmine. Moreover, the supplemental inclusion of 3,4 DAP, ephedrine, or albuterol often yields significant clinical improvement. RAPSN has been shown to interact with KHDRBS1.

Dok-7 is a non-catalytic cytoplasmic adaptor protein that is expressed specifically in muscle and is essential for the formation of neuromuscular synapses. Further, Dok-7 contains pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domains that are critical for Dok-7 function. Finally, mutations in Dok-7 are commonly found in patients with limb-girdle congenital myasthenia.

Dok-7 regulates neuromuscular synapse formation by activating MuSK

The formation of neuromuscular synapses requires the muscle-specific receptor tyrosine kinase (MuSK). In mice genetically mutant for MuSK, acetylcholine receptors (AChRs) fail to cluster and motor neurons fail to differentiate. Because Dok-7 mutant mice are indistinguishable from MuSK mutant mice, these observations suggest Dok-7 might regulate MuSK activation. Indeed, Dok-7 binds phosphorylated MuSK and activates MuSK in purified protein preparations and in muscle in-vivo by transgenic overexpression. Furthermore, the nerve-derived organizing factor agrin fails to stimulate MuSK activation in muscle cells genetically null for Dok-7. Thus, Dok-7 is both necessary and sufficient for the activation of MuSK.

The requirement for MuSK in the formation of the NMJ was primarily demonstrated by mouse “knockout” studies. In mice which are deficient for either agrin or MuSK, the neuromuscular junction does not form.

Upon activation by its ligand agrin, MuSK signals via the proteins called Dok-7 and rapsyn, to induce “clustering” of acetylcholine receptors (AChR). Cell signaling downstream of MuSK requires Dok-7. Mice which lack this protein fail to develop endplates. Further, forced expression of Dok-7 induces the tyrosine phosphorylation, and thus the activation of MuSK. Dok-7 interacts with MuSK by way of protein “domain” called a “PTB domain.”

In addition to the AChR, MuSK, and Dok-7 other proteins are then gathered, to form the endplate to the neuromuscular junction. The nerve terminates onto the endplate, forming the neuromuscular junction—a structure which is required to transmit nerve impulses to the muscle, and thus initiating muscle contraction.

Homozygous mutation of Dok-7 is responsible for a form of congenital myasthenic syndrome (CMS) that is unique among disorders in this category because it affects muscles in the limbs and trunk but mostly spares the face, eyes, and functions of the mouth and pharynx (chewing, swallowing and speech). Salbutamol can be effective in relieving CMS symptoms attributable to Dok-7 mutations.

Structure

There are three potential 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.

  • McDonnell KM, Grow WA (2004). “Reduced glycosaminoglycan sulfation diminishes the agrin signal transduction pathway”. Developmental Neuroscience. 26 (1): 1–10. doi:10.1159/000080706. PMID 15509893. S2CID 42558266.

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

Functions

Agrin may play an important role in the basement membrane of the microvasculature as well as in synaptic plasticity. Also, agrin may be involved in blood–brain barrier (BBB) formation and/or function and it influences Aβ homeostasis.

Research

Agrin is investigated in relation with osteoarthritis. In addition, by its ability to activate the Hippo signaling pathway, agrin is emerging as a key proteoglycan in the tumor microenvironment.

Clinical significance

AGRN gene mutation leads to congenital myasthenic syndromes and myasthenia gravis.

A recent genome-wide association study (GWAS) has found that genetic variations in AGRN are associated with late-onset sporadic Alzheimer’s disease (LOAD). These genetic variations alter β-amyloid homeostasis contributing to its accumulation and plaque formation.

References

  1. GRCh38: Ensembl release 89: ENSG00000188157 – Ensembl, May 2017
  2. GRCm38: Ensembl release 89: ENSMUSG00000041936 – Ensembl, May 2017
  3. “Human PubMed Reference:”. National Center for Biotechnology Information, U.S. National Library of Medicine.
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Further reading

External links

Protein: nerve tissue protein
Signaling pathway: Wnt signaling pathway
Protein kinases: tyrosine kinases (EC 2.7.10)
Enzymes

Categories: 

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