A number of regulatory enzymes have a serine (or sometimes a tyrosine or threonine) residue at a regulatory site. This can undergo phosphorylation catalysed by a protein kinase, as shown in Figure 10.5. Phosphorylation may increase or decrease the activity of the enzyme. Later, the phosphate group is removed from the enzyme by phosphoprotein phosphatase, thus restoring the enzyme to its original state. These responses are not instantaneous, but they are rapid, with a maximum response within a few seconds of hormone stimulation.
The reduction in activity of pyruvate dehydrogenase in response to increased concentrations of acetyl CoA and NADH (section 10.5.2) is the result of phosphorylation. This control of enzyme phosphorylation by substrates is unusual. In most cases, the activities of protein kinases and phosphoprotein phosphatases are regulated by second messengers released intracellularly in response to fast-acting hormones binding to receptors at the cell surface. 5'-AMP, formed by the action of adenylate kinase (section 10.2.2.1) also activates a protein kinase — in this case it is acting as an intracellular messenger in response to changes in ATP availability, rather than in response to an external stimulus.
The hormonal regulation of glycogen synthesis and utilization is one of the best understood of such mechanisms. Two enzymes are involved, and obviously it is not desirable that both enzymes should be active at the same time:
In response to insulin (secreted in the fed state) there is increased synthesis of glycogen and inactivation of glycogen phosphorylase. In response to glucagon (secreted
in the fasting state) or adrenaline (secreted in response to fear or fright) there is inactivation of glycogen synthase and activation of glycogen phosphorylase, permitting utilization of glycogen reserves. As shown in Figure 10.6, both effects are mediated by protein phosphorylation and dephosphorylation:
There is a further measure of instantaneous control by intracellular metabolites which can over-ride this hormonal regulation:
response to glucagon and adrenaline
active glycogen synthase
Inactive (phosphorylated) glycogen synthase inactive active (phosphorylated)
glycogen Phosphorylase glycogen Phosphorylase active glycogen synthase phosphate glycogen Phosphorylase glycogen Phosphorylase phosphate phosphate phosphate response to insulin
Figure 10.6 Hormonal regulation of glycogen synthetase and glycogen phosphorylase — responses to glucagon or adrenaline and insulin.
10.3.1 MEMBRANE RECEPTORS AND G-PROTEINS
A cell will respond to a fast-acting hormone only if it has cell-surface receptors that bind the hormone. The receptors are transmembrane proteins; at the outer face of the membrane they have a site which binds the hormone, in the same way as an enzyme binds its substrate, by non-covalent equilibrium binding.
When the receptor binds the hormone, it undergoes a conformational change that permits it to interact with proteins at the inner face of the membrane. These are known as G-proteins because they bind guanine nucleotides (GDP or GTP). They function to transmit information from an occupied membrane receptor protein to an intracellular effector, which in turn leads to the release into the cytosol of a second messenger, ultimately resulting in the activation of protein kinases.
The G-proteins that are important in hormone responses consist of three subunits, a, P and Y As shown in Figure 10.7, in the resting state the subunits are separate, and the a-subunit binds GDP When the receptor at the outer face of the membrane is occupied by its hormone, it undergoes a conformational change and recruits the a-, P- and Y-subunits to form a G-protein trimer—receptor complex. The complex then reacts with GTP, which displaces the bound GDP Once GTP has bound, the complex dissociates.
The a-subunit of the G-protein with GTP bound then binds to, and activates, the effector, which may be adenylyl cyclase (section 10.3.2), phospholipase C (section 10.3.3) or an ion transport channel in a cell membrane, resulting in release of a second messenger.
The a-subunit slowly catalyses hydrolysis of its bound GTP to GDP As this occurs, the a-subunit—effector complex dissociates, and the effector loses its activity. The G-protein subunits are then available to be recruited by another receptor that has been activated by binding the hormone.
10.3.2 Cyclic amp and cyclic GMP as second messengers
One of the intracellular effectors that is activated by the (G-protein a-subunit)—GTP complex is adenylyl cyclase. This is an integral membrane protein which catalyses the formation of cyclic AMP (cAMP) from ATP (Figure 10.8). cAMP then acts as the second messenger in response to hormones such as glucagon and adrenaline. It is an allosteric activator of protein kinases. cAMP is also formed in the same way in response to a number of neurotransmitters.
As shown in Figure 10.8, phosphodiesterase catalyses the hydrolysis of cAMP to yield 5'-AMP, thus providing a mechanism for termination of the intracellular response to the hormone. Under normal conditions, 5'-AMP is then phosphorylated to ADP by the reaction of adenylate kinase — it is only under conditions of relatively low ATP
availability and relatively high ADP that adenylate kinase acts to form 5'-AMP as an intracellular signal (section 10.2.2.1).
Phosphodiesterase is activated in response to insulin action (which thus acts to terminate the actions of glucagon and adrenaline), and is inhibited by drugs such as caffeine and theophylline, which therefore potentiate hormone and neurotransmitter action.
In the same way as cAMP is formed from ATP by adenylyl cyclase, the guanine analogue, cGMP, can be formed from GTP by guanylyl cyclase. This may be either an integral membrane protein, like adenylyl cyclase, or a cytosolic protein. cGMP is produced in response to a number of neurotransmitters and also nitric oxide, the endothelium-derived relaxation factor that is important in vasodilatation.
The active (G-protein a-subunit)—GTP released in response to binding of 1 mol of hormone to the cell-surface receptor will activate adenylyl cyclase or guanylyl cyclase for as long as it contains GTP The hydrolysis to yield inactive (G-protein a-subunit)— GDP occurs only relatively slowly. Therefore, a single molecule of (G-protein a-subunit)-GTP will lead to the production of many thousands of mol of cAMP or cGMP as second messenger.
There is an equilibrium between cAMP or cGMP bound to protein kinase and in free solution in the cytosol and therefore accessible to phosphodiesterase for inactivation. Each molecule of cAMP or cGMP activates a molecule of protein kinase for as long as it remains bound, resulting in the phosphorylation of many molecules of target protein.
Each enzyme molecule that has been activated by protein kinase will catalyse the reaction of many thousands of mol of substrate per second until it is dephosphorylated by phosphoprotein phosphatase.
10.3.3 Inositol trisphosphate and diacylglycerol as second messengers
The other response to G-protein activation involves phosphatidylinositol, one of the phospholipids in cell membranes (section 126.96.36.199). As shown in Figure 10.9, phos-phatidylinositol can undergo two phosphorylations, catalysed by phosphatidylinositol kinase, to yield phosphatidylinositol bisphosphate (PIP2). PIP2 is a substrate for phospholipase C, which is activated by the binding of (G-protein a-subunit)—GTP The products of phospholipase C action are inositol trisphosphate (IP3) and diacylglycerol, both of which act as intracellular second messengers.
Inositol trisphosphate opens a calcium transport channel in the membrane of the endoplasmic reticulum. This leads to an influx of calcium from storage in the endoplasmic reticulum and a 10-fold increase in the cytosolic concentration of calcium ions. Calmodulin is a small calcium-binding protein found in all cells. Its affinity for calcium is such that at the resting concentration of calcium in the cytosol (of the order of 0.1 ^mol/L), little or none is bound to calmodulin. When the cytosolic concentration of calcium rises to about 1 ^mol/L, as occurs in response to opening of the endoplasmic reticulum calcium transport channel, calmodulin binds 4 mol of calcium per mol of protein. When this occurs, calmodulin undergoes a conformational change, and calcium—calmodulin binds to, and activates, cytosolic protein kinases, which, in turn, phosphorylate target enzymes.
The diacylglycerol released by phospholipase C action remains in the membrane, where it activates a membrane-bound protein kinase. It may also diffuse into the cytosol, where it enhances the binding of calcium—calmodulin to cytosolic protein kinase.
Inositol trisphosphate is inactivated by further phosphorylation to inositol tetrakisphosphate (IP4), and the diacyl glycerol is inactivated by hydrolysis to glycerol and fatty acids.
The active (G-protein a-subunit)—GTP released in response to binding of 1 mol of hormone to the cell-surface receptor will activate phospholipase C for as long as it contains GTP, and therefore, a single molecule of (G-protein a-subunit)—GTP complex will lead to the production of many thousands of mol of IP3 and diacylglycerol as second messengers.
Each molecule of diacylglycerol will activate membrane protein kinase until it is hydrolysed (relatively slowly) by lipase, so resulting in the phosphorylation of many molecules of target protein, each of which will catalyse the metabolism of many thousands of mol of substrate per second, until it is dephosphorylated by phosphoprotein phosphatase.
Similarly, each molecule of IP will continue to keep the endoplasmic reticulum calcium channel open until it is phosphorylated to inactive inositol tetrakisphosphate, thus maintaining a flow of calcium ions into the cytosol. Each molecule of calcium— calmodulin will bind to, and activate, a molecule of protein kinase for as long as the cytosol calcium concentration remains high. It is only as the calcium is pumped back into the endoplasmic reticulum that the calcium concentration falls low enough for calmodulin to lose its bound calcium and be inactivated. Again each molecule of phosphorylated enzyme will catalyse the metabolism of many thousands of mol of substrate per second, until it is dephosphorylated by phosphoprotein phosphatase.
The insulin receptor itself is a protein kinase, which phosphorylates susceptible tyrosine residues in proteins. When insulin binds to the external part of the receptor, there is a conformational change in the whole of the protein, resulting in activation of the protein kinase region at the inner face of the membrane. This phosphorylates, and activates, cytosolic protein kinases, which, in turn, phosphorylate target enzymes, including phosphoprotein phosphatase (see Figure 10.6), cAMP phosphodiesterase (see Figure 10.8) and acetyl CoA carboxylase.
There is amplification of the response to insulin. As long as insulin remains bound to the receptor, the intracellular tyrosine kinase is active, phosphorylating, and therefore activating, many molecules of protein kinase, each of which phosphorylates many molecules of target enzyme.
A number of the receptors for growth factors have the same type of intracellular tyrosine kinase as does the insulin receptor. A mutant of the receptor for the epidermal growth factor (EGF) is permanently activated, even when not occupied by EGF. This results in continuous signalling for cell division and is one of the underlying mechanisms in cancer.
Was this article helpful?
Statistics For Obesity Are Rising And The Majority Of People Are Not Getting Enough Exercise Nor Are Having Any Regard For Their Health! Will You Finally Make Good On Your Promise And Set Your Goals To Improve Your Fitness And Live The Healthy Lifestyle You Want? With A Little Bit of Motivation, You Can Set Yourself On the Correct Path To Losing Weight And Feeling Great Using Nothing But Purely Natural Means!