Structural Biochemistry/Membrane Proteins/G Protein Coupled Receptor
G Protein Coupled Receptor (GPCR)
[edit | edit source]G protein coupled receptors have seven transmembrane spans(TM), an amino-terminus facing outwards and a carboxy terminus facing the interior of the cell. Evidence and studies have shown that TM5 and TM6 play a more important part in the course of activation. The G protein is inside the cell, and it binds to either GTP or GDP; GTP is the same as ATP except that it has a G (guanine) base nucleotide instead of an A (adenine) nucleotide. When the G protein is bound to GTP, it activates, but when the G protein is bound to GDP, it deactivates; in other words, when there is a signaling molecule the G protein will bind to GTP, but if there is no signaling molecule, the G protein will bind to GDP.
The GPCR is a huge family of proteins that is involved in sight (rod cells have rhodopsin as a GPCR, which bind to a photon of light--a signaling molecule), in smell (olfactory receptors bind to different smells, and there are specific receptors for specific smells), and in taste (sweet, bitter, and umami are GPCR while salty and sour are ion coupled).
Structure of GPCRs
[edit | edit source]Besides the seven transmembrane helices, GPCRs vary a lot in structure. Unfortunately, it is very difficult to actually form spatial models of different GPCRs because the GPCRs have the ability to assume several conformations. Thus, during the course of crystallization, one has to minimize the conformation changes throughout so as to attain reasonable levels of visualization of the crystals. Only a few structures of several GPCRs have been proposed.
The structural differences of GPCRs are responsible for the differences in their specificity towards different G-proteins.
Epinephrin Signaling
[edit | edit source]Epinephrin signaling stimulates glycogen and breaks it down. First, epinephrin binds to GPCR, which causes the GPCR to recruit a G protein. This G protein drops its GDP and associates with GTP instead. The G protein then activates production of cAMP (a molecule). As a result, cAMP activates a protein called PKA (protein kinase A). PKA phosphorylates enzymes and activates molecules, which results in the breakdown of glycogen to glucose. PKA can also phosphorylate enzymes to inactivate and convert glucose to glycogen.
In this process, the first messenger is epinephrine while the second messenger is cAMP (the second messenger is a molecule that is produced). Amplification, the hallmark of pathways, is when one epinephrine produces multiple cAMPs to activate PKA.
The pathway can be turned off by doing one of the following: removing the epinephrine, removing cAMP, getting rid of GPCR (this is feasible through endocytosis), hydrolyzing GTP to GDP to deactivate the G protein, or by dephosphorylizing PKA targets to deactivate phosphates by removing them.
Cholera toxin inhibits the switch from GTP to GDP, which does not allow the pathway to be turned off. This increases cAMP production, which then increases ion secretion. This is dangerous because water molecules follow the ions that are secreted out, which can lead to dehydration and eventual death.
Examples of GPCRs
[edit | edit source]Rhodopsin
[edit | edit source]Rhodopsin belongs to the class-A receptors of GPCRs and it was through rhodopsin that scientists were able to find the common structure of the seven transmembrane helixes in GPCRs. Rhodopsin has a covalently bound ligand--retinal. Retinal has several different forms; this is the property that can help in structural studies of GPCR, setting it apart from most GPCRs. In the absence of light, retinal exists in 11-cis form. This form is inactive in G protein transducin and rhodopsin containing retinal in this form is called dark rhodopsin. In the presence of light, retinal, through isomerization, changes into its all-trans form. When this happens, through a series of conformational changes, it is activated into metarhodopsin II.
From the study of its structure, ionic locks were discovered in its structure. Ionic lock is basically a collection of hydrogen bonds and ionic interactions that were formed between the residues of rhodopsin. This is responsible for the complete inactivity of dark rhodopsin and its G protein, transducin.
Its three-dimensional structure was proposed in year 2000 but it is still continually being refined.
β-adrenergic receptors (β-ARs)
[edit | edit source]β-adrenergic receptors are class-A GPCRs that are bound to diffusible ligands.
Crystal structure of β-adrenergic receptors
[edit | edit source]The structure of β-AR were studied using two methods
- GPCR crystals are usually formed by the interactions that take place with its water soluble, exterior parts. Thus, the main strategy is to provide a larger water-soluble surface area for crystals to form on. Therefore, β-AR was made to complex with fragment antigen-binding(Fab). A Fab fragment was prepared from an antigen that could identify TM5 and TM6 β-AR; Fab bound to the receptor, but does not affect β-AR's ability to bind with its ligand. The GPCR-Fab complex was then used for crystallization. The result was partially successful; the interior side formed reasonably well but the exterior side did not.
- The other way was to substitute the third intracellular loop(ICL3) between TM5 and TM6 with T4 lysozyme(T4L). T4L was chosen because of the past successes it had with crystallization and its length; it had similar length to ICL3. This will not only increase the water-soluble surface area, but it will also stabilize β-AR. β-AR with T4L has the same affinity for both ligands and inverse ligands. The unaltered β-AR binds 2-3 times more to its ligand than to its inverse ligand. The altered β-AR produced better crystals; Most of it could be visualized, except for the last 71 residues facing the interior of the cell.
In both methods, the inverse ligand, carazolol was used to stabilize its conformation.
References
[edit | edit source]"Biology" by Neil A. Campbell and Jane B. Reece
Weis, William I; Kobilka, Brian K (2008), "Structural Insights into G-protein-coupled receptor activation", Current Opinion in Structural Biology, doi:10.1016/j.sbi.2008.09.010, PMID 18957321 {{citation}}: Unknown parameter |acess date= ignored (|access-date= suggested) (help)