G-protein-linked receptors activate G proteins by reducing the strength of GDP b

Question

G-protein-linked receptors activate G proteins by reducing the strength of GDP binding. This results in rapid dissociation of abound GDP, which is then replaced by GTP, which is present in the cytosol in much higher concentrations than GDP. What consequences would result from a mutation in the alpha subunit of the G protein that caused its affinity for GDP to be reduced without significantly changing its affinity for GTP? Compare the effects of this mutation with the effects of the cholera toxin.

Explanation / Answer

Activiated G Proteins bind to enzymes or other proteins and alter the target protein’s activity. G Proteins are guanine-nucleotide binding proteins. G Protein-linked Receptors have an extracellular N-terminus and a cytosolic C-terminus separated by seven transmembrane alpha helices connected by peptide loops. One of the extracellular segments has an unique messenger-binding site. The cytosolic loop between the 5th and 6th alpha helices specifically binds a particular G protein. G Proteins bound to GTP are active, those bound to GDP are not. The two classes of G Proteins are large heterotrimeric G Proteins and small monomeric G Proteins. In heterotrimeric G proteins (G alpha, beta, gamma), when a messenger binds the G Protein-linked receptor, the receptor changes conformation to allow association of the trimeric G Protein with the receptor. G-alpha subunit binds the guanine nucleotide (GDP or GTP). This interaction causes the G alpha subunit to release the GDP, pick up a GTP and detach from the complex. Depending upon the G protein in question, either the GTP-G alpha complex, the G beta- G gamma complex or both bind target protein(s). The G alpha will remain an activating messenger until the GTP is hydrolyzed by the G alpha subunit (GTP -> GDP +Pi). The "inactive" GDP-G alpha will then reassociate with the G-beta-G alpha complex to rapidly turn down this pathway when the original stimulatory signal is removed. Large numbers of G proteins provide diversity for signal transduction events. Some bind potassium or calcium ion channels in neurotransmitters. Some activate kinases (enyzmes that phosphorylate). Some cause either the release or formation of major second messengers such as cyclic AMP (cAMP) and calcium ions. cyclic AMP is a second messenger used by a major class of G proteins. cyclic AMP (cAMP) is generated by adenylyl cyclase which is embedded in the plasma membrane with the enzymatic activity in the cytoplasm. Adenylyl cyclase is activated by binding an activated alpha subunit of the Gs G-protein (GTP-Gs). Phosphodiesterase continally degrades cAMP so in the absence of the ligand and active G-Protein, cAMP levels are reduced. Protein kinase A (PKA), a cAMP-dependent kinase, is the main intracellular target of cAMP. PKA phosphorylates a number of proteins that bear the key short stretch of amino acids, the PKA phosphorylation site (PKA PO4 site). PKA transfers a phosphate from the ATP to a serine or threonine in the PKA PO4 site. cAMP activates the catalytic subunits by causing the release of the negative regulatory subunits. Disruption of G Protein signaling causes several human diseases. Vibrio cholerae (causes cholera) secretes the cholera toxin which alters salt and fluid in the intestine normally controlled by hormones that activate Gs G-Protein to increase cAMP. The cholera toxin enzymatically changes Gs so that it is unable to convert GTP to GDP. Gs can not then be inactivated and cAMP levels remain high causing intestinal cell to secrete salt and water. Eventually dehydration can lead to death (cholera). Many G proteins use inositol triphosphate and diacylglycerol as second messengers G Protein-linked Receptor. The Gp G-Protein is activated by a ligand binding its G Protein-linked receptor to activate phospholipase C. Phosphatidylinositol-4,5-bisphosphate (PIP2) is cleaved by phospholipase C into two molecules cytosolic inositol-1,4,5-triphosphate (InsP3) and membrane-bound diacylglycerol (DAG). The InsP3 receptor, a ligand-gated calcium channel in the endoplasmic reticulum, binds InsP3 and calcium ions are released into the cytosol. Calcium binds a protein known as calmodulin, and the Ca-calmodulin complex act to activate an number of processes. DAG remains membrane-bound and activates protein kinase C (PKC). Cholera toxin binds Ga The enzymatic activity of cholera toxin ADP-ribosylates the G-protein alpha Some portion of the cholera toxin subunit, thus, blocking its reassociation penetrates the membrane with GTP..Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate

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