Structural Biochemistry/Cell Signaling Pathways/G-Proteins and G-Protein Coupled Receptors

G-Proteins and G-Protein Coupled Receptors
G-Protein coupled receptors (GPCRs) are a group of seven transmembrane proteins which bind signal molecules outside the cell, transduct the signal into the cell and finally cause a cellular response. The GPCRs work with the help of a G-Protein which binds to the energy rich GTP.

Also known as heptahelical receptors, serpentine receptors, and G protein-linked receptors. These proteins make up transmembrane receptors whose purpose is to find molecules on the outside of the cell and initiate the signal transduction pathways. The signal transduction pathways are the processes by which a cell changes the form of one signal into the stimulus or a signal of another. These processes are carried out by enzymes. As the number of proteins and molecules increases, the size of the signal cascade increases rapidly, allowing for a large response, to a relatively small initiation factor.

G protein linked receptors are activated by ligands in the form of hormones, proteins, or other signaling molecule. This in turn leads to the activation of an intracellular G-protein by way of a certain interaction with the receptor. The G proteins act like relay batons to pass messages from circulating hormones into cells and transmit the signal throughout the cell with the ultimate goal of amplifying the signal in order to produce a cell response. Firstly, a hormone such as an epinephrine encounters a receptor in the membrane of a cell then a G protein is activated as it makes contact with the receptor to which the hormone is attached. Lastly, the G protein passes the message of a hormone to the cell by switching on a cell enzyme that triggers a response (Medicines by Design 46).

In addition to signaling, they have other physiological roles: -Sense of smell-the olfactory epithelium receptors bind odorants and pheromones -Mood regulation-receptors in the brain bind neurotransmitters (dopamine) -Immune system regulation- deals with inflammation and response to foreign bodies -Nervous system transmission-proteins control blood pressure, heart rate, and digestive processes -Cell density sensing

Another example of G-protein-coupled receptors (GPCRs) in the body has to do with the sense of taste. Five different tastes have been recognized by science: sweet, salty, sour, bitter and umami. Umami, the flavor of meaty foods, was the taste most recently added to the list, as it was identified by a Japanese scientist in 1908. GPCR taste receptors are found on our taste buds, and there are several different types.
 * The receptors associated with the umami taste are given the titles T1R1 and T1R3. These are class C GPCRs, which means they have an N-terminus outside of the cell.  In this case, these receptors’ extracellular domain is quite large and the shape of the active site contains two halves; such domains have been dubbed Venus flytrap (VFT) domains due to this characteristic shape.  The active site of the VFT of the T1R1 and T1R3 receptors binds amino acids, especially aspartate and glutamate (two amino acids with one and two CH2 groups in its R chain, respectively).
 * Sweet tastes are recognized by receptors in the same family as the umami tastes: T1R2 and T1R3. The VFT domains on these GPCRs have many active sites that can bond many ligands.  This is why these particular receptors can not only recognize carbohydrates, like sugars, but also some types of amino acids, peptides, proteins, and the ligands of artificial sweeteners.
 * While the sweet and umami tastes are recognized by class C receptors, the unpalatable bitter sensation is acknowledged by class A GPCRs, which do not have the large N-terminus of the class C. There are more than 30 different types of GPCRs for the bitter taste, dubbed the T2R receptors.  Like the T1R3 sweet/umami receptor, these T2Rs can each detect many chemicals with the bitter taste.  Compared to the two or three types of GPCRs for the sweet and umami tastes, thirty different kinds seems like an unnecessary amount.  The explanation, however, seems to be one of evolution.  Bitter tastes are associated with toxins or poisons produced by plants and insects, so those mammals that recognized the vile taste and no longer consumed the toxin had the opportunity to procreate.

G-protein coupled receptors are only found in eukaryotic cells and are found in a variety of sizes.

There are two main pathways that the G-proteins follow. The first is the cAMP pathway, and the second is the Phosphatidylinositol signal pathway.

The cAMP pathway has five players that aid in the signal transduction: a hormone that stimulates the receptors, a regulative g protein, Adenylyl cyclase, protein kinase A, and cAMP. The stimulative hormone is a receptor that binds with the stimulative signal molecules. The g-proteins is linked to the simulative hormone receptor and its alpha subunit can stimulate activity. Adenylyl cyclase is a transmembrane glycoprotein that catalyzes ATP to form cAMP with the help of a cofactor, usually Magnesium or Manganese ions. Protein kinase A is an enzyme used for cell metabolism. The cAMP transfers the effects of hormones which are unable to pass directly through the cellular membrane. It also regulates the effects of adrenaline and glucagon in addition to regulating the passage of Calcium ions through the ion channels. cAMP activates PKA (protein kinase A) which is usually an inactive tetramer. cAMP binds to the regulatory subunits of the kinase and causes the dissociation of the regulatory and catalytic subunits. This dissociation activates the catalytic units and allows them to phosphorylate the substrate proteins.

The phosphatidylinositol signal pathway has the signal molecule bind with the G-protein receptor. This activates the phospholipase C which is located in the membrane. Lipase hydrolyzes phosphatidylinositol in to two messengers with bind with the receptors in the membrane. This opens up a Calcium ion channel and activate protein kinase C which causes the cascade of signals.

Studies have shown that GPCR consists of transcriptional, post-transcriptional and post-translational mechanisms and recently, it has been observed that by splicing the pre-mRNA regulates GPCR activity by targeting GPCR Secretin of the exon on a 14 amino acid sequence.

Mechanism
Inside the cell, on the plasma membrane, G Protein binds GDP when inactive and GTP when active. When the GPCRs binds to a signal molecule, the receptor is activated and changes shape, thereby allowing it to bind to an inactive G Protein. When this occurs, GTP displaces GDP which activates the G Protein. The newly activated G Protein then migrates along the cell membrane until it binds to an enzyme and changes the enzyme's shape and conformation. This change in the enzyme structure leads to the next step in the pathway and generates a cellular response. After transduction, G Protein functions as a GTPase and hydrolyzes the bound GTP which causes a phosphate group to fall off. This regenerates GDP and inactivates the G Protein and the cycle repeats.

Receptor Properties

 * 1) Receptors are proteins located on either the extracellular or intracellular of the cell
 * 2) Receptors are highly specific for ligand.
 * 3) Receptors form a complex with the ligand.
 * 4) Receptors have an equilibrium constant for both the forward and reverse reaction, (K1 and K2, respectively).
 * 5) The concentration of the ligands and the receptors set the amplitude of the responses. More hormones and receptors will yield a stronger response.
 * 6) Receptors can dimerize to increase their activity.

Membrane-Bound Receptors (Cell Surface Receptors)
Membrane-bound receptors are peptide hormone receptor. The membrane-bound receptors are proteins located on the plasma membrane. The receptors can be single polypeptide chains or have up to four subunits. Some may have up to seven transmembrane domains. Some hormones that these receptors bind to are prostaglandins, ACTH, glucogon, catecholamines, parathyroid hormone, etc. The receptor initiates signal transductions when a hormone is bind to it. Many of the signal transductions involve second messengers. Second messengers are compounds that are activated or produced when the receptors bind to the ligands. The second messengers are normally cyclic adenosine monophosphate (cAMP) molecules or cyclic guanine monophosphate (cGMP) molecules, Ca2+, or protein kinase C (PKC). During the signal transduction, many compounds form complexes and either activate or inactivate other molecules inside the cell. These reactions are coupled with G-proteins. Other receptors used are tyrosine kinase, serine kinase, and guanyl cyclase domains known as Enzyme-Linked receptors.

Seven-transmembrane domain receptor has three cytoplasmic loops: loops I, II, and III. Loop III and the carboxyl tail end have a kinase activity, which enables them to autophosphorylate. This receptor is coupled with Guanine Nucleotide Binding Protein (G-Protein). G-protein is a heterotrimeric, meaning it has three non-identical subunits (alpha, beta, and gamma subunits). The alpha subunit has a GDP bound to it when inactive and has a GTP bound when active.

Forms of G-Protein
The alpha subunit has a GDP bound to it when inactive and has a GTP bound when active.

G-Protein Coupled Receptor Structure
The structure of most G-Protein Coupled Receptors are not very well known. The typical method of determining protein structure is by x-ray crystallography of the protein once it has been crystallized. However, due to the membrane environment, flexibility, and dynamic shifting of GPCRs, it is difficult to form crystals of them. Some have been crystallized by mutating certain amino acids to stabilize the structure, but there is no universal way to study them all.

Although it is tough to find exact information about the structure there are a few known traits of the proteins. G-proteins are integral membrane proteins that make up transmembrane helices. The parts of the receptor can be glycosylated. Of the seven transmembrane proteins, each one may or may not contain an ion channel. These glycosylated loops are made of two cysteine residues that form disulfide bonds which help stabilize the receptor structure.

Conformational change

The receptor molecule exists in equilibrium between the active and inactive states. The ligand binding pushes the equilibrium towards the active sites. There are three types of ligands that bind to the g-proteins. The first are agonists, ligands that shift the equilibrium towards the active states. Inverse agonist shift the equilibrium towards the inactive states, and neutral antagonists are ligands that do not change the equilibrium.

Uses of G-Protein Coupled Receptors
G-Protein Coupled Receptors are widespread in their use. In the eyes, Opsins, a GPCRs, translate electromagnetic radiation into cellular signals thus allowing visual perception. In the nose, olfactory epithelium binds odorants and pheromones which allow for the sense of smell. However, there are problems associated with GPCRs. Many human diseases, including bacterial infections, involve the GPCRs where bacteria produce toxins which interfere with the function of the G-Protein. Examples of such diseases include cholera, whooping cough, botulism, etc.

Here is a closer view of how altered G-protein affects cholera and whooping cough. When there is a β subunit binding to Gαs gangliosides and a catalytic subunit entering the cell, then choleragen, a toxin resulted from cholera, forms. The catalytic subunit alters the Gαs ganglioside in which α part of the protein is adjusted via attaching ADP-ribose to arginine. As a result of this alteration, Gαs ganglioside become stable, meaning that now Gαs ganglioside is in its active form. The active Gαs ganglioside then does its job to activate protein kinase A, denoted PKA. The chloride channel is by PKA, PKA enters and thus there is no more absorption of Na+. This is saying that there is a huge loss of NaCl and water in the body as seen in the symptoms of cholera. In treating cholera, the most effective way is to rehydrate the body using glucose-electrolyte solution.

Whooping cough, on the other hand, is different from cholera in which the ADP ribose moiety is added by the toxin. In this case, Ca 2+ chanel is closed whereas the K + channel is opened by Gαs ganglioside. The result is that the Gαs ganglioside is in its inactive form and ultimately ending up with uncontrollable coughs.

In addition, Pharmacologists estimate that 60% of all medicines achieve their effect by acting on G Protein pathways. Since G protein is a switch molecule which passes the message inward (like relay baton), it can be turned on only when needed, then shut off. Some illnesses like cholera, occur when a G protein is errantly left on. Discovery about Gprotein switches and its structure which is made into 3 subunits (alpha, beta, gamma) will help us understand how we can inhibit the transmission or increase with some other ligand. G-protein coupled receptors are trans-membrane receptor proteins that when activated by ligands (hormones, proteins or other signaling molecules), they lead to the activation of an intracellular G-protein through a specific interaction with the receptor. The G-protein in turn transmits the signal to other proteins within the cell to ultimately amplify the signal and produce a cellular response. Understanding the structure and dynamics of the receptor could clarify the specific interactions the receptor makes with the ligand on the outside and the Gprotein on the intracellular side, thereby leading to the understanding of how the receptor works. Consequently, drugs (agonists or antagonists) can be designed to bind the receptor and control its response, whether it's to transmit the signal to the G-protein or inhibit the transduction. Moreover, monitoring the effects of such signals can help in understanding the type of induced cellular responses and potentially uncover diseases that are proliferated in this manner.

Reference
Berg, Jeremy "Biochemistry", Chapter 14 Signal-Transduction Pathways. pp 421-422. Seventh edition. Freeman and Company, 2010.