Collybistin

The gene ARHGEF9 (aka ARHDH) codes for Collybistin. ARHGEF9 can be found in various regions of the brain, such as the cerbral cortex, hippocampus and cerebellum, but only in locations where neurological synapse fire.There are three domains in the structure of collybistin;^[4] the src-homology 3 (SH3) domain, the dbl-homology (DH) domain, and the pleckstrin homology (PH) domain. The SH3 domain, located at the N-terminus, binds to the regulatory sequences and prevents the protein from staying active.^[5] SH3 acts as the regulator within the collybistin structure since most activity depends on the interactions with other proteins at the SH3 domain. Similarly, Without the PH domain, the protein is rendered useless as it cannot complete the plasma membrane targeting and clustering of gephrin.^[4] The DH domain is responsible for interacting and binding with GTPases.^[6] The DH domain regulates binding with gephrin.

Collybistin is categorized as a modular protein structure.^[7][8] The protein has the ability of rearranging its conformation to be more efficient in its activity, or folding certain domains to inhibit protein activity.

Collybistin is responsible for the gephyrin-dependent clustering of GABA receptors in the brain. ARHGEF9 functions to organize synapes and inhibit them when necessary. Additionally, ARHGEF9 is responsible guanine nucleotide exchange factors (GEFs).^[9] Through immunohistochemistry^[10] it was discovered that the production of collybistin changes throughout developmental stages of organisms. Any mutations within this gene may cause various negative symptoms within the organism. The organism may experience things such as intellectual disabilities, anxiety,^[11] hyperekplexia,^[12] etc. In a study done in 2011,^[13] there was a direct link found between a nonsynonymous deletion in the ARHGEF9 gene and mental disability, along with physical disability. Similarly, it has been reported that missense mutations within the gene have also caused mental and physical disabilities to the organism with the mutated gene.

Pentylenetetrazol (PTZ)^[14] induced seizures severely damage a wide range of synapes in the brain, including collybistin. However, when collybistin is overexpressed in the brain, it has the ability to prevent PTZ- induced seizures and protects the neurotransmitter pathways in the brain. If collybistin is not overexpressed and falls victim to PTZ, then gephrin is not clustered and synapes are not stabilized. Additionally, the inhibitory receptors become weakened, which may cause the seizure to become stronger and life-threatening.

Similarly, a missense mutation of the gene, referred to as a R290H gene variant,^[15] was recently discovered as a cause of epilepsy in human patients. This mutation alters the communication within the protein, specifically the folding of the DH and PH domains. Improper folding may lead to degradation or for the cell to be targeted by the ubiquitin proteasome pathway^[16] for termination. This mutation causes the protein to become weaker and prevents it from binding to other proteins, such as gephyrin, and inhibits plasma membrane connection. Similar to the PTZ-induced seizures, the R290H gene variant weakens the inhibitory receptors and prevents the development of the synapse. Due to activity of regulatory inhibition being reduced, the neurons become overactive, which leads to epilespy and other mental disabilities.

Without collybistin, there is a lack of proper regulator positioning,^[17] synapse organization, and inhibition signalling. Additionally, collybistin is responsible for synaptic scaffolding and cytoskeleton organization.^[18] With specific binding to the a2 subunit, collybistin is crucial for the proper function of sites that tell nuerons when to fire. In a 2018 study, a mutated collybistin protein lead to an increase in mortality rates in mice within the first 20 days after birth. The mice that faced early mortality had a disconnection in the neuropathways and receptors between collybistin and GABA_A receptors. The signalling from the GABA receptors became weaker and decayed at a more rapid rate. The animals suffer from spontaneous seizures that increased in strength each time. As seizure strength increased, there was a larger disruption in brain function and breathing. The seizures were identified as the ultimate cause of death, while a decline in the proper function of collybistin was found to be the proximate cause of death.

There are currently 3 known isoforms of Collybistin. Each isoform is similar in that they contain a RhoGEF binding (DH) domain, and a pleckstrin homology (PH) domain.^[19] Where they differ is at the N-terminus in both sequence and whether or not a Src-homology (SH3) domain will be present. The SH3 group, which is responsible for the inhibition of protein activity, is connected to the N-Terminus of Collybistin.^[20] However, the isoform connected to the SH3- part of this terminus are constantly active and continuously signal to other proteins.^[21] This region is able to cluster gephrin without being instructed to do so by neurotransmitters or other proteins. The next isoform to discuss for Collybistin is connected to the SH3+ end of the N-Terminus. This isoform will remain folded until it is required for clustering. Similarly, the last isoform also remains folded until neuroligin-2 or neuroligin-4 bind to the protein.^[22] They also differ in the C-terminus sequence. The isoforms are referred to as CB1, CB2, and CB3. These three forms have been identified in rats, while only CB3 has been identified in humans and is referred to as hPEM2.^[23]

Splice variants are the same proteins structurally, but they contain different mRNA sequences produced by alternative splicing. Splice variants of collybistin retain the DH and PH domains, however they dffer in the SH3 groups of the N-terminal and the C-terminal domain.^[24] SH3- variants are able to induce translocation to submembrane locations due to them being enzymatically active. The splice variant most commonly found in the central nervous system is SH3+. This splice variant is autoinhibited. The SH3+ and the SH3- variants both increase the production of gephyrin clustering. SH3+ does so by producing non-synaptic clusters that do not have fractions. Whereas, the SH3- variant focuses on the gephyrin clusters at postsynaptic sites. Additionally the SH3- variant is able to form ternary complexes, but SH3+ is unable to. Although these variants are able to increase the levels of gephyrin clustering, they also directly impact GABAergic synapse formation. Additionally, these any muattions related to these variants result in cognitive impairments.