Cell Surface Membrane And Receptor Assembly Biology Essay

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The number of cells on cell surface membrane is determined by the rate of receptor assembly and maturation through the secretary pathway and plasma membrane insertion and also the rates of receptor recycling between the surface and intracellelluar regions

The number of receptors found on the cell surface is also dependent on the relationship between inhibitory proteins, adhesion proteins and other components that partake in the synaptic inhibition control. [1] 

During the process of membrane trafficking formation, assembly and transfer of GABAaR to the cell surface and degradation process have been controlled by a group of proteins which have shown to interact with the GABAaR.

These proteins regulate GABAaR trafficking by interacting with the GABAA RECEPTOR. They play an important role in the transportation and development of GABAA RECEPTOR as well as internalization, recycling and inhibiting the receptor from proteasomal degradation

Formation of the GABAA RECEPTOR occurs in the ER (endoplasmic reticulum). Oligomerisation (process of the formation of single subunits into hetrometric receptor) occurs where the N-terminus of the α , β and γ subunits are assembles into the 5 part channel; GABAA RECEPTOR. [2] 

Various studies on the oligomerisation of receptors have shown that if a single subunits is expressed alone than it does not leaves the ER, instead it is rapidly broken down (degradation) but if the α and β subunit are expressed alongside one another with a site for GABA to bind, than it becomes a 'functional' GABAA RECEPTOR. [3] 

Receptors are also sensitive to other constituents such as benzodiazepines, so receptors also have as part of their structure a γ2 subunit which is essential for 'synaptic targeting' [4] As mentioned earlier the most common GABAA RECEPTOR is made up of 2α, 2β and 1γ. [5] 

Proteins

For the regulation and maintenance of the cell surface receptors, a number of proteins are present in cell to aid reaction pathways of receptor life cycle. It begins in the ER, in the presence of two chaperone protein molecules, namely BiP and Calnexin. [6] the cell attempts to limit the amount of diversity in the GABAA RECEPTORs that are expressed on the cell surface through selective oligomerisation [7] and quality control. The N- terminus of specific subunits hold a preferential sequence, where by the formation of a receptor from that particular subunit is prioritized, forming a carefully regulated range of receptors with the aid of BiP and Calnexin.

Another protein whose role is to aid in post-Golgi GABAA RECEPTOR trafficking is BIG2. It catalyses the reaction of GDP to GTP and studies have shown that this protein interacts with GABAA RECEPTORs intracellular regions of β 1, 2 and 3 subunits. [8] 

This protein is found primarily in the Golgi network and around post synaptic sites. [9] where studies have shown that expression of BIG2 with β subunits showed a marked increase in the number of receptors that left the ER., thus an increased chance of GABAA RECEPTOR reaching the cell surface membrane. [10] studies have demonstrated BIG2 contribute in endocytosis of GABAA RECEPTOR and endocytic sorting of GABAA RECEPTOR, but this is still to be verified.

17KDa GABAA RECEPTOR associated protein; also known as GABARAP, acts together with the subunits of GABAA RECEPTOR. [11] It binds to microtubules and NSF and does this in vitro and in vivo. [12] This protein can be found primarily in the Golgi apparatus alongside GABAA RECEPTOR. [13] Absence of GABARAP in GABAergic synapse [14] would indicate its role in the cell as specifically transporting GABAA RECEPTOR, thus over expression of GABARAP with GABAA RECEPTOR has shown a positive correlation in cell surface GABAA RECEPTOR expression due to increases trafficking [15] . This process can be abolished by a mutation which corrupts the addition of phospholipids to GABARAP, increasing membrane association so critical for the control of GABAA RECEPTOR trafficking by GABARAP.

GABARAP works alongside a handful of other proteins in order to increase cell surface expression of GABAA RECEPTORs. N-ethylmaleimide sensitive factor (also known as NMF) works directly alongside GABARAP in order to regulate GABAA RECEPTOR trafficking [16] . They interact to promote forward trafficking of GABAA RECEPTOR from Golgi apparatus [17] . NSF has a binding affinity for GABAA RECEPTORs β subunit. Studies have shown, over expression of NSF in a cell, causes a decrease in GABAA RECEPTOR cell surface expression [18] , this is the opposite effect of over expression of GABARAP. [19] Together these two proteins, when co-expressed reduce the number of receptors found at the cell surface. [20] These findings could suggest that NSF has other roles in the endocytic pathways.

GABARAP also works alongside PRIP-1. PRIP-1 and PRIP2 are found in mammals. PRIP-1 is found predominantly in the brain and PRIP-2 expressed ubiquitously [21] . These PRIP proteins compete against GABARAP for γ2 subunit binding. Recent studies have shown PRIP can form a bridge across GABARAP and receptor, this helping to traffic receptors contacining γ2 subunits to the synaptic membrane. [22] Studies on PRIP 1 and 2 double knockout mice showed impaired γ2 trafficking. PRIP 1 and 2is also involved in regulation of receptor trafficking by regulation phosphorylation [23] .

GODZ also known as Golgi-specific DHHC zinc finger domain protein, also regulates trafficking by binding γ2 subunits by mediating palmitoyl acyl transfer to these subunits [24] . Palmitoylation is where a saturated fatty acid palmitate covalently attaches to a protein. Studies have proven to play part in trafficking and function at inhibitory and excitory synapses. [25] Cys-cysteine repeat domains of GODZ, thus makes it ideal to efficiently palmitoylate the γ2 subunit.

GODZ is found in trans-Golgi network and plays a very important role in reducing the build up of GABAA RECEPTORs containing γ2 subunits in synapses. [26] Thus the primary role of GODZ protein is to control the trafficking of secretary pathway and delivery to the plasma membrane. [27] 

Once cells have matured and surfaced by the aid of proteins, it is equally important to regulate receptor internalisation. Internalisation is also known as clathrin-dependent endocytosis. Whereby cells 'internalise', by the inward budding of the plasma membrane into a clathrin coated vesicle under the cell surface. Studies have suggested that internalisation of GABAA RECEPTORs is ny the protein Clathrin and dynamin dependent mechanism (as mentioned above)

Clathrin and synamin proteins have been shown to be responsible for these protein dependent mechanisms, leading to receptor internalisation. [28] (although there has been cases of receptor internalisation. [29] )

The clathrin adaptor protein (AP2) structure encompasses a µ2 subunit which is responsible for the interactions with GABAA RECEPTOR β subunits through an atypical basic patch binding motif [30] . In order to aid receptor into endocytic pathway, thus binding of AP2 and β subunits encompasses a key site for phosphorylation by serine and threonine kinases [31] . (AP2 receptor interactions is actually negatively regulated by derene phosphorylation.)

GABAA RECEPTORs γ subunit has also shown a high affinity to the binding of Clathrins µ2 subunit which occurs through tyrosine type motif (made up of amino acids and a bulky hydrophobic side chain) in the γ subunit.

As well as AP2 and GABAA RECEPTOR interactions GABARAP have shown to interact with Clathrin heavy chanins and PRIP-1 protein with AP2. [32] 

Once receptors are internalised by aid of clathrin and dynamin protein, two mechanism pathways can occur either the GABAA RECEPTOR will be degrades by lysosome (lysomoal degregation) or recycling where receptors will rapidly be transported back to the cell surface. [33] 

Again proteins play a role in regulation which receptors are degregaded and which will be recycled. Huntington associated protein (HAP1) inhibits GABAA RECEPTORs to be selected for lysosomal degradation instead promoting recycling of cell to cell surface membrane. [34] Studies in animals where HAP1 protein levels were reduced showed a correlating decrease in GABAA RECEPTOR activity and change in animal behaviour. These findings are indicative of the importance of HAP1 in the normal behaviour of animals. [35] 

These proteins all play an important role in regulation the number of cell surface receptor, by assisting GABAA RECEPTOR assembly, maturation, internalization, degradation and recycling.

Other proteins that play a role in these are also present, they are not as significant as those previously discussed but still play a role in the regulation of cell surface receptors.

GRIF-1 is a protein whose specific role is unclear at the moment, but has shown to interact with potassium channels Kir2.1 and GABAA RECEPTOR in vitro to regulate cell receptor trafficking. [36] 

TRAK-1, another protein has also been seen to interact with these GABAA RECEPTOR complexes. [37] 

Both these proteins through studies have shown to traffic organelles within the cell including mitochondria and endosomes. [38] 

As mentioned previously GABARAP binds to microtubules in the cell, another protein namely gephyrin also binds to tubules within the cell. These two may therefore be responsible in binding of GABAA RECEPTORs to microtubules. Gephyrin acts as a scaffolding protein giving receptors stability at the synapse. [39] 

Phosphorylation of GABA is.

GABAA RECEPTOR is a substrate for a few protein kinases and phosphates. Proteins are also involved in regulating these reaction mechanisms. The receptor for activated C kinase (RACK1) in addition to protein kinase C (PKC) and also PKA, also take part in GABAA RECEPTOR. [40] Proteins used also in dephosphorylation of receptors also interact with β subunits protein phosphatises PP1 and PP2.

Phosphorylation

Regulation of GABAA RECEPTOR also ocur from phosphorylation of the receptor. The β subunits of receptors encompass which phosphorylation can take place, there are serine residues which are phosphorylated by kinases (incl. PKA and PKC). The process of phosphorylation increase number of GABAA RECEPTOR on the cell surface of the cell surface and increases trafficking to these sites by phosphorylating β2 subunit of the GABAA RECEPTOR. [41] 

Brain derived neurotrophic factors have also been seen to correlate increase in receptor function on a number of cell surface membranes. [42] Regions of β subunits which partake in interacting with clathrin adapter AP2 are also regions that are vital for β subunit phosphorylation [43] which in turn has a negative regulation of AP2 binding.

The clathrin adaptor protein AP2, mentioned how binding motif increased receptor number entering endocytic pathway. Dephosphorylation of S408/S409 on the β3 subunits is responsible in unmasking this binding patch motif in order to enhance the endocytosis of receptor

Alongside the β subunit, γ2 asksi acts as a substrate in phosphorylation process by serine and PKC. Dephosphorylation of γ2 subunit has shown to cause a decrease in GABAA RECEPTOR function. Most γ2 subunits are significantly phosphorylated [44] , which causes an interaction between the clathrin AP2 and γ2 subunit (via specific tyrosine motif) to be inhibited and reducing its high affinity to bind to AP2, thus reducing the number of receptors entering the endocytic pathway. But in cell conditions where dephosphorylation occurs, there could be an increase in number of cells entering endocytic pathway [45] .

The GABAA RECEPTOR receptors interact with clathrin AP2 protein, with their β and γ subunits to allow phosphorylation to occur through 2 different kinase pathways (tyrosine kinases for γ2 subunits and serine/threonine for β subunits) [46] 

Palmitoylation plays an important role in regulating transport of receptors to the cell surface. [47] The subunit of the receptor γ2 contains cysteine residues whose role is to act as a substrate for palmitoylation. Importance of palmitoylation was demonstrated in a study whereby cysteine residues in γ2 were all mutated (palmitoylation could not occur as the substrate was mutated), and in a separate study palmitoylation inhibitor 2-BrP was applied to neurones; both these resulted in a vast reduction on GABAA RECEPTOR clusters at membrane cell surface [48] 

GODZ protein previously shown to prevent the build up of GABAA RECEPTOR at membrane, also mediates palmitoylation of the γ2 subunits on receptors, important in normal functioning of the inhibitory synapses.

Ubiquitination is a post translational modification of a receptor by covalently binding one or more ubiquitin monomers, thus labelling it for protosomal degradation. GABAA RECEPTOR β3 subunit contains a number of lyseine residues which act as a substrate for ubiquitination [49] . In a study where all 12 lysine's were mutated in their intracellular loop of the β3 subunit, there was a marked decrease in the levels of β3 ubiquitination thus increase in cell surface expression. [50] 

Ubiquitination is dependent on neuronal activity so where neuronal activity is inhibited, ubiquitination increases dramatically, thus causing a decrease in cell surface expression and vice versa if neuronal activity was to be increase cell surface receptor expression will increase. Ubiquitination of unassembled subunits within the endoplasmic reticulum are subject to increase rate of ubiquitination and are thus degrades by proteomes. [51] This process also plays significant role in stability of receptors and degradation. In the case of epilepsy, a mutation found in A322D of GABAA RECEPTOR α1 subunit leads to subunit misfolding, then ubiquination, followed by rapid degradation through the protosome system, thus leading to a decreased expression of the receptors at cell surface, leading to inhibitory effects, thus epilepsy.

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