Mechanisms of Hormonal Action Bryant Miles

Transcription

1 Mechanisms of ormonal Action Bryant Miles Multicellular organisms need to coordinate metabolic activities. Complex signaling systems have evolved using chemicals called hormones to regulate cellular activities. Animals have an endocrine systems consisting of glands that release hormones into the blood stream where the hormones are carried through the body where they reversibly bind to their target receptors. Mechanisms of ormone Action 1. If the substrate concentration is rate limiting, then hormones may alter the concentration of the substrate to increase or decrease the rate of flux. 2. ormones may promote the reversible phosphorylation of the flux controlling enzymes to change the conformation of the enzyme s active site either activating or deactivating the enzyme. 3. ormones may promote the dephosphorylation of the flux controlling enzymes. 4. ormones can affect the concentrations of allosteric effectors. 5. ormones can induce or repress genes to change the amount of enzyme present in the cell. We have already studied the peptide hormones insulin and glucagons, and the catecholamines: epinephrine and norepinephrine. These hormones bind to receptors of the target cells. These hormones have high affinity for their target receptors with KD values ranging from to 10-6 M. nly a minute amount of the hormones are required to induce response. The binding of the hormone stimulates a chemical activity that is communicated into the cell. Steroids are another type of hormone. Steroids are derived from cholesterol and regulate metabolism, electrolyte balance, inflammatory responses and sexual function. Steroid hormones occasionally bind to cellular receptors to induce their effects. Steroids are lipids. That means they are permeable to biological membranes. Most steroids passively diffuse into the nucleus where the bind to transcription factors directly regulating genetic expression of genes. ormones provide a mechanism to maintain homeostasis, to respond to changing metabolic conditions and to regulate cellular differentiation and genetic expression. Signal transduction begins when a hormone is released into the blood stream. The hormone is the called the first messenger. The hormone binds to specific cell membrane receptors. The information that the signal molecules are present must be transmitted across the cell membrane. The binding of the hormone to the receptor drives conformational changes that produce a response in the cytostolic side of the cell. The response results in the change of concentration of small molecules called the second messengers in the inside of the cell. Second messengers include cam, cgm, Ca 2+, inositol 1,4,5 triphosphate (I 3 ) and diacylglycerol, DAG.

2 These second messengers can diffuse to cellular compartments, such as the nucleus where the second messenger influences gene expression. The signal carried by the hormone becomes amplified, as one hormone bound to the receptor can instigate the formation of 100 s of second messenger molecules. ne common response to a second messenger is the activation of protein kinases which use AT to phosphorylate serine, threonine and tyrosine residues of the target enzymes. This phosphorylation is reversible due to protein phosphatases which are enzymes that remove the phosphoryl groups from the serine, threonine and tyrosine residues. Eventually the signal needs to be terminated. therwise the cells would lose responsiveness to new signals. Three types of hormone receptors. 1. The 7-transmembrane segment (7-TMS) receptors which are integral membrane proteins with seven transmembrane α helical segments. Examples are the G-binding proteins. 2. The single transmembrane segment (1-TMS) catalytic receptors which are proteins that a single transmembrane α helix that spans the membrane. Examples are tyrosine kinases and guanylate cyclases. 3. ligomeric ion channels which consist of multiple protein subunits. These channels are also called ligand gated channels because the bind of the hormone to the receptor opens the ion channel.

3 7-TMS Receptors. 7-TMS receptors are involved in transmitting information initial by signals such as photons of light, odorants, hormones and neurotransmitters. So far 7,000 7-TMS receptors have been discovered and the list is rapidly growing. ne example, is the β adrenergic receptor which binds epinephrine aka adrenaline the fight or flight hormone. The binding of epinephrine to this receptor located on the outside of the cell induces a conformational change that is detected inside of the cell. The binding of epinephrine activates a G protein. The activated G protein in turn activates adenylyl cyclase which converts AT into cam and pyrophosphate. G roteins G-proteins are intermediaries in signal transduction from 7-TMS receptors. They are called G-proteins because they containing binding sites for guanosine nucleotides. In the case of the β-adrenergic receptor, the resting G-protein is a heterotrimer consisting of α, β and γ subunits. The α subunit contains the guanosine nucleotide binding site. In the resting state, the α subunit has GD bound and associates with receptors such as the glucagon receptor or the β-adrenergic receptor. The binding of the hormone to the receptor produces allosteric conformational changes that cause the α subunit to release GD and bind GT. The binding of GT is a switch which causes the α subunit to dissociate from the G βγ dimer. The GT bound α subunit diffuses laterally through the membrane until is associates with adenylate cyclase. The association of these two proteins activates adenylate cyclase which then starts producing cam. A single hormone bound to its receptor can activate 100s of G α molecules Adenylate Cyclase

4 2 - hosphodiesterase C C 3 Second messengers need to have short half lives so that the response can be rapidly terminated. hosphodiesterase hydrolyzes the phosphodiester bond to convert cam to AM. This is the enzyme that is inhibited by caffeine one of my favorite biomolecules. Caffeine increases the cellular concentration of cam by inhibiting phosphodiesterase. The G proteins ultimately need to reset themselves. The G α subunit has an C 3 intrinsic GTase activity. The bound GT will slowly be hydrolyzed into GD Caffeine and i. This GTase activity is like a built in clock that spontaneously resets the G α subunit after a short period of time. After the G α subunit has hydrolyzed GT it tightly binds the GD. When the G α subunit had GD bound it dissociates from adenylate cyclase turning this enzyme off and reassociates with the G βγ dimer to reform the heterotrimer. This requires the hormone to be bound to the receptor to keep adenylate cyclase active. Every 7-TMS receptor has a G type protein associated with it. They do not all function through the same G-protein used by the glucagon and β-adrenergic receptors. The typical G-proteins are heterotimers consisting of α, β and γ subunits in the resting state. The binding of the hormone to the receptor causes the exchange of GD for GT in the α subunit. The G α subunit then dissociates from the G βγ dimer and associates with an effector protein. Eventually the G α subunit hydrolyzes the GT, binds the GD tightly, dissociates from the effector protein and reassociates with the G βγ dimer to reform the heterotrimer. Glucagon and epinephrine bind to different receptors yet activate the same G protein which in turn activates the same effector protein, adenylate kinase. ther effector proteins activated by other G proteins are phospholipase C, phospholipase A2, potassium channels, sodium channels, calcium channels. There are more than 20 different G-proteins discovered to date. A few are listed to the left. The hormone-receptor mediated processes regulated by G proteins may be stimulatory as in the example of the epinephrine, β-adrenergic receptor, or inhibitory. Each G-protein interacts with a stimulatory G-protein denoted G αs or with an inhibitory G protein denoted G αi. Epinephrine also binds to a α-adrenergic receptor. The α-adrenergic receptor associates with a G αi protein. The binding of epinephrine to the a- adrenergic receptor causes the exchange of GD for GT causing the G αi to dissociate from the G βγi

5 dimer. The inhibition comes from either the G αi subunit associated with adenylate cyclase to directly inhibit the cyclase, or by the action of G βγi which associates with the G αs subunit when it has GT bound. The G αi thus competes with adenylate cyclase for G αs. Cholera Toxin Cholera is a gram positive bacterium that causes cholera. Both the cholera bacteria and the toxin remain localized in the intestinal epithelial cells. Cholera causes severe diarrhea in its victims which often leads to death due to severe dehydration. The cholera toxin is protein that catalyzes the AD-ribosylation of Arg-201 of the G αs subunit we have been talking about. This cholera toxin uses AD + as a substrate to produce the AD-ribosylated Arg-201. The AD-ribosylation of Arg-201 destroys the GTase activity of the G αs subunit. The G-protein becomes locked into the active state producing prolonged activation of adenylate cyclase. The elevated levels of cam cause the intestinal epithelial cells to secrete high volumes of fluid.

6 The ertussis Toxin Bordetella pertussis is the bacterium that causes whooping cough. Bordetella pertussis produces a toxin which is an enzyme that catalyzes the AD-ribosylation of a Cysteine residue of the G αi protein. The AD-ribosylated G αi protein cannot exchange GD for GT which prevents G αi from dissociating from the G αβγi heterotrimer. G αi then cannot inhibit adenylate cyclase. ertussis is a systemic infection affecting the regulation of adenylate cyclase throughout the entire body. hosphatidyl Inositol Bisphosphate Cyclic AM is not the only second messenger produced by 7-TMS receptors and the associated G- proteins. Let s examine another second messenger cascade that is induced by many hormones to evoke a number of cellular responses, the phosphinositide cascade. Vasopressin is an antidiuretic hormone that binds to the vasopressin receptor which is yet another 7-TMS receptor. The binding of vasopressin to the receptor induces the associated G protein to exchange GD for GT causing the G αq subunit to dissociate from the G βγq dimer. The G αq subunit with GT bound associates with phospholipase C activating the lipase. The activated lipase hydrolyzes the phosphodiester bond linking the phosphorylated inositol to the diacylglycerol. The cleavage of this phosphodiester bond produces 2 second messengers, inositol 1,4,5- triphosphate (I 3 ) and diacylglycerol (DAG). DAG diffuses laterally in the lipid membrane while I 3 is water soluble and diffuses into the cytosol of the cell.

7 Inositol 1,4,5 triphosphate is a second messenger that binds to I 3 -gated channels located in the membrane of the endoplasmic reticulum and the calcisomes. The binding of I 3 promotes the rapid release of Calcium ions. Calcium ions are yet another second messenger which triggers muscle contractions in muscle tissue, exocytosis and glycogen breakdown. Inositol 1,4,5 triphosphate has a short half life. It is rapidly converted into derivatives such as inositol or inositol 1,3,4,5 tetrakisphosphate that do not open the calcium channels. Lithium is used to treat bipolar disorders. Lithium inhibits the recycling of inositol 1,3,4 triphosphate.. Diacylglycerol (DAG) is another second messenger. Diacylglycerol activates a number of protein effectors. We will concentrate on one, protein kinase C (KC). This is a kinase that uses AT to phosphorylate serine and tyrosine residues of many target proteins. Before it is activated by DAG, this protein is free in the cytosol. A portion of the C1A subunit has a sequence that is very similar to the sequence of the target proteins. This pseudosubstrate sequence occupies the active site of the enzyme inactivating it. When phosphatidylinositol diphosphate is hydrolyzed by the activated phospholipase C, DAG is generated in the lipid membrane. KC binds to the DAG creating an attachment of KC to the membrane. When CK is anchored to the membrane the psuedosubstrate sequence interacts with head groups of the phospholipids of the membrane. This activates the kinase activity of this enzyme. KC also requires Calcium ions for activity (remember that I3 induces the release of Ca 2+ from the endoplasmic reticulum and the calciosomes). The two second messengers DAG and I3 work in tandem to activate KC. DAG also has a short half life. It is phosphorylated to form phosphatidic acid or it is hydrolyzed to its glycerol and fatty acid components.

8 Calcium ions are another important second messenger. The binding of certain hormones and signal molecules to receptors in the plasma membrane can cause transient increases in cytoplasmic Ca 2+ levels. Increases in cytoplasmic calcium levels activate a wide variety of enzymatic processes. Cyclic AM activates the opening of plasma Ca 2+ channels allowing extracellular calcium to stream in. Within the cell there are calcium reservoirs such as the endoplasmic reticulum and the calciosomes. The internal stores are not released by cam. They only respond to I3, the second messenger derived from phosphatidyl inositol. There are a number of intracellular calcium binding proteins which in turn regulate many cellular processes. The most common is calmodulin shown to the left. Some others are paravalbumin, troponin C and the annexin proteins. There are over 170 calcium modulated proteins known. Most of these have the characteristic EF hand. The conformation of these EF hand proteins changes dramatically when calcium is bound. Shown below are the conformational changes that occur when calmodulin binds 4 calcium ions. When Calmodulin has 4 calcium ions bound, it is bound by target proteins which activate them. ne example is calmodulin dependent protein kinases (CaM kinases). These kinases phosphorylate many proteins that regulate fuel metabolism, ionic permeability, neurotransmitter synthesis and neurotransmitter release. These kinases are inactive until they bind calmodulin which has 4 calcium ions bound.