“Describe the signalling pathways downstream of the heterotrimeric G proteins Gs, Gi and Gq”
Heterotrimeric guanine nucleotide-binding proteins (G proteins) comprise a family of membrane proteins that are present in all eukaryotic cells and are responsible for controlling metabolic, neural, and developmental functions. The proteins are bound to the inner surface of the cell membrane and act as signal transducers that communicate signals from various hormones, neurotransmitters, chemokines and local mediators. G proteins become activated when members of large superfamily of receptors called G protein-coupled receptors receive those extracellular signals. The signals are then sent to several distinct intracellular signalling pathways. The interaction of these pathways with one another forms a network which regulates a broad range of cellular processes.
Heterotrimeric G proteins consist of three distinct parts: Gα subunit, Gβ and Gγ, the latter forming a tightly associated Gβγ heterodimer. The Gα subunit has an enzymatic (GTPase) activity and catalyses the conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). The hydrolysis of GTP regulates the length of the signal. The Gα protein can be divided into four subunits according to the sequence identity and functional similarity: Gαs, Gαi, Gαq, Gαo. The different subfamilies of Gα subunit show selectivity with respect to both the receptors and the effectors with which they couple, having specific recognition domains in their structure complementary to specific G protein-binding domains in the receptor and effector molecules. The beta-gamma complex is also a vital part of the heterotrimeric G protein. When it is bound to Gα subunit, it increases the affinity of Gα subunit for GDP. This causes the G protein to be in the inactive state. Once it is separated from the Gα subunit it can also participate in its own signalling pathways.
In the resting state, the G protein exists as an αβγ trimer, which may or may not be precoupled to the receptor with GDP occupying the site of the α subunit. G protein signalling cycle begins by binding of the agonist molecule to a GPCR. The binding causes the GPCR to become activated and a conformational change occurs. In this new conformational state the receptor binds to the G-protein complex in such a way that the G-protein is then able to undergo its own conformational change. The interaction of the trimer with the receptor causes the release of the bound GDP molecule, which is immediately replaced with GTP molecule (GDP-GTP exchange). The binding of GTP causes the dissociation of the G protein trimer, releasing α-GTP and βγ subunits. In this separated form the subunits diffuse in a membrane and are able to associate with various enzymes and ion channels causing the activation of the target. Association of α or βγ subunits with target enzymes or ion channels can cause either activation or inhibition depending on which G protein is involved. G protein activation results in amplification because a single agonist-receptor complex can activate several G protein molecules in turn, and each of these can remain associated with the effector enzyme for long enough to produce many molecules of the product. The product is often a second messenger (such as cyclic AMP) and further amplification occurs before the final cellular response is produced. The activation of the effector molecules lasts until the G-protein alpha subunit catalyses the conversion of GTP to GDP. When G-alpha subunit has GDP bound, it dissociates from the effector and reunites with βγ, completing the cycle.
G proteins play a vital role in the transduction of the signals from a wide range of extracellular agents such as the hormones, neurotransmitters, chemokines or local mediators. These agents signal to the main G protein families to regulate metabolic enzymes, ion channels or transcriptional regulators. For example, the Gs and Gi signalling pathways regulate the cyclic AMP whereas the Gq protein pathway is responsible for regulating the inositol 1,4,5-trisphosphate (IP3).
Gs protein family has a very well defined effector pathway called the adenylyl cyclase pathway, also known as cAMP-dependent pathway. It is a signalling cascade triggered by a G protein-coupled receptor and used in cell communication. The Gs alpha subunit of the stimulated G protein complex exchanges GDP to GTP and is then released from the complex. In a cAMP-dependent pathway, the enzyme called adenylyl cyclase is activated by the Gs alpha subunit. The enzyme catalyses the conversion of ATP into the second messenger cAMP. The role of cAMP is significant because it mediates various cell responses such as the increase in heart rate, cortisol secretion and the breakdown of glycogen and fat. This pathway can also activate enzymes and regulate gene expression. The increase in concentration of cAMP may lead to the activation of the enzyme called protein kinase A (PKA). Protein kinases regulate various functions of cellular proteins by controlling the protein phosphorylation. Protein kinases are responsible for processes such as glycogen and fat metabolism or the increased contraction of the heart.
The action of the epinephrine (adrenaline) on skeletal muscle illustrates the role of G proteins in the regulation of cyclic AMP very well. Glucose is stored in skeletal muscle in the form of glycogen. During vigorous exercise, ATP is required to fuel muscle contraction and this requires the breakdown of glycogen to glucose. Epinephrine which is secreted into the blood from the adrenal medulla is responsible for that process. Increased levels of adrenaline activate an adrenergic receptor on the muscle membrane called beta-adrenoceptor. The receptors are linked to Gs and when an alpha subunit of Gs dissociates, it activates adenylyl cyclase. The activation of adenylyl cyclase leads to an increase in the intracellular concentration of cyclic AMP. The cyclic AMP activates protein kinase A which then breaks glycogen down to glucose.
It is vital that the cAMP-dependent pathway is controlled, otherwise it can ultimately lead to hyper-proliferation, which may contribute to the development of cancer. Any alterations in number, structure and function of the receptors lead to the disorder in cellular signal transduction which result in various diseases. One of the examples is hyperthyroidism – a condition in which the thyroid gland produces and secretes excessive amounts of thyroid hormones. The thyrotropin receptor is a member of G protein-coupled receptors family and is activated by the thyrotropin-releasing hormone (TRH). TRH stimulates the secretion of thyroid-stimulating hormone (TSH) which is coupled to the Gs protein. Thus, the effects of TSH are largely mediated by the stimulation of adenylyl cyclase and cyclic AMP concentrations. Mutation in TSHR gene leads to the hyperactivity of the cAMP pathway which results in the hyper-activation of a gland. Another example of a disease caused by the disorder in cellular signal transduction is cholera. Cholera is an infection of the small intestine caused by the bacterium Vibrio cholerae. Cholera toxin binds to the intestinal cells (enterocytes) when it is released from the bacteria in the intestine. As soon as the toxin enters the cell it activates the Gs protein through ADP-ribosylation reaction. As a result the G protein is locked in its GTP-bound form which continually stimulates adenylyl cyclase to produce cAMP. Increased Gs activation leads to the increased adenylyl cyclase activity which in turn increases the intracellular concentration of cAMP to more than 100-fold over normal and over-activates cytosolic PKA. The activated PKA then phosphorylate the CFTR chloride channel proteins which leads to the efflux of chloride ions. This leads to secretion of H2O, Na+, K+, and HCO3- into the intestinal lumen. The process results in a rapid fluid loss from the intestine, leading to severe dehydration.
While Gs activates adenylyl cyclase and stimulates the production of cyclic AMP, the Gi protein inhibits adenylyl cyclase. Activation of receptors coupled to Gi causes the intracellular level of cyclic AMP to fall. This is how somatostatin inhibits the release of gastrin by the G cells of the gastric mucosa. Gi mainly inhibits the cAMP dependent pathway by inhibiting adenylyl cyclase activity, decreasing the production of cAMP from ATP, which, in turn, results in decreased activity of cAMP-dependent protein kinase. When Gi receptors get activated, they release activated G protein beta-gamma subunits from inactive heterotrimeric G protein complexes. G beta-gamma dimeric protein interacts with GIRK channels and opens them so that they become permeable to potassium ions, resulting in hyperpolarisation of the cell and thus termination of the action potential. In chronic atrial fibrillation there is an increase in this inwardly rectifying current because of constantly activated by IK,Ac channels. Increase in the current results in shorter action potential duration experienced in chronic atrial fibrillation and leads to the subsequent fibrillating of the cardiac muscle. These receptors are primarily found in the heart as well as in the brain.
The last family of G-alpha subunit is the Gq protein which activates phosphoinositide-specific phospholipase C isoenzymes (PLC-beta). The isoenzymes are activated by heterotrimeric G-proteins and GPCRs by either the release of alpha-subunits of the Gq family or by G beta-gamma dimers from activated Gi family members. These isoenzymes catalyse the hydrolysis of the phospholipid phosphatidylinositol bisphosphate (PIP2) to release second messengers IP3 and DAG. The second messengers function as intracellular mediators and both increase intracellular concentrations of free calcium ions. Gq has also been reported to activate the transcription factor oNFkB through proline-rich tyrosine kinase-2. The process of stimulation of the second messenger concentration increase is initiated by the release of acetylcholine which acts on a muscarinic receptor. This receptor activation leads to the dissociation of the G-proteins and the subsetuent activation of phospholipase C.
DAG generated by the hydrolysis of phosphatide inositol is able to diffuse in the plane of the membrane where it progresses to activate the enzyme protein kinase C (PKC) to phosphorylate targeted proteins in generating various physiological responses such as increasing the rate of DNA transcription or receptor activation whilst IP3 releases calcium from internal stores, leading to the activation of calcium-dependent events such as secretion.
To conclude, G-proteins play a crucial role in many signalling tasks and regulate an incredible range of bodily functions.