Mechanisms of Signal Transductio1

8/17/2019 Mechanisms of Signal Transductio1

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Mechanisms of Hormonal ActionBryant Miles

Multicellular organisms need to coordinate metabolic activities. Complex signaling systems have evolvedusing chemicals called hormones to regulate cellular activities. Animals have an endocrine systemsconsisting 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 Hormone Action1. 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. Hormones 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. Hormones may promote the dephosphorylation of the flux controlling enzymes.4. Hormones can affect the concentrations of allosteric effectors.5. Hormones 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 andnorepinephrine. These hormones bind to receptors of the targetcells. These hormones have high affinity for their targetreceptors with KD values ranging from 10 -12 to 10 -6 M. Only aminute amount of the hormones are required to induceresponse. The binding of the hormone stimulates a chemicalactivity that is communicated into the cell. Steroids areanother type of hormone. Steroids are derived from cholesteroland regulate metabolism, electrolyte balance, inflammatoryresponses and sexual function. Steroid hormones occasionally

bind to cellular receptors to induce their effects. Steroids are

lipids. That means they are permeable to biologicalmembranes. Most steroids passively diffuse into the nucleuswhere the bind to transcription factors directly regulatinggenetic expression of genes.

Hormones provide a mechanism to maintain homeostasis, to respond to changing metabolic conditionsand 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 thesignal 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 inthe inside of the cell. Second messengers include cAMP, cGMP, Ca 2+, inositol 1,4,5 triphosphate (IP 3)and diacylglycerol, DAG.


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These second messengers can diffuse to cellular compartments, such as the nucleus where the secondmessenger influences gene expression. The signal carried by the hormone becomes amplified, as onehormone bound to the receptor can instigate the formation of 100’s of second messenger molecules.

One common response to a second messenger is the activation of protein kinases which use ATP to

phosphorylate serine, threonine and tyrosine residues of the target enzymes. This phosphorylation isreversible due to protein phosphatases which are enzymes that remove the phosphoryl groups from theserine, threonine and tyrosine residues.

Eventually the signal needs to be terminated. Otherwise the cells would lose responsiveness to newsignals.

Three types of hormone receptors.

1. The 7-transmembrane segment (7-TMS) receptors which are integral membrane proteins withseven transmembrane α− helical segments. Examples are the G-binding proteins.

2. The single transmembrane segment (1-TMS) catalytic receptors which are proteins that a singletransmembrane α− helix that spans the membrane. Examples are tyrosine kinases and guanylatecyclases.

3. Oligomeric ion channels which consist of multiple protein subunits. These channels are alsocalled ligand gated channels because the bind of the hormone to the receptor opens the ionchannel.


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7-TMS Receptors.

7-TMS receptors are involved in transmitting information initial by signalssuch as photons of light, odorants, hormones and neurotransmitters. So far7,000 7-TMS receptors have been discovered and the list is rapidly growing.One example, is the β−adrenergic receptor which binds epinephrine akaadrenaline the fight or flight hormone. The binding of epinephrine to thisreceptor located on the outside of the cell induces a conformational changethat is detected inside of the cell. The binding of epinephrine activates a G

protein. The activated G protein in turn activates adenylyl cyclase whichconverts ATP into cAMP and pyrophosphate.

G ProteinsG-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 theguanosine nucleotide binding site. In the resting state, the α subunit has GDP bound and associates withreceptors such as the glucagon receptor or the β-adrenergic receptor. The binding of the hormone to thereceptor produces allosteric conformational changes that cause the α subunit to release GDP and bindGTP. The binding of GTP is a switch which causes the α subunit to dissociate from the G βγ dimer. TheGTP 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 cAMP. Asingle hormone bound to its receptor can activate 100s of G α molecules.

Adenylate Cyclase

N

NN

N

NH 2

O

OHHO

HH

HH

OPO

O-

O

P

O

O -P

O

O -

-O O

N

NN

N

NH 2

O

OHO

HH

HH

O

P

O-

O


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Second messengers need to have short half lives so that the response can berapidly terminated. Phosphodiesterase hydrolyzes the phosphodiester bond toconvert cAMP to AMP. This is the enzyme that is inhibited by caffeine one ofmy favorite biomolecules. Caffeine increases the cellular concentration ofcAMP by inhibiting phosphodiesterase.

The G proteins ultimately need to reset themselves. The G α subunit has anintrinsic GTPase activity. The bound GTP will slowly be hydrolyzed into GDPand Pi. This GTPase activity is like a built in clock that spontaneously resetsthe G α subunit after a short period of time. After the G α subunit has

hydrolyzed GTP it tightly binds the GDP. When the G α subunit had GDP bound it dissociates fromadenylate 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 sameG-protein used by the glucagon and β-adrenergic receptors. The typical G-proteins are heterotimersconsisting of α , β and γ subunits in the resting state. The binding of the hormone to the receptor causesthe exchange of GDP for GTP in the α subunit. The G α subunit then dissociates from the G βγ dimer andassociates with an effector protein. Eventually the G α subunit hydrolyzes the GTP, binds the GDP 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 turnactivates the same effector protein, adenylate kinase. Other effector proteins activated by other G proteins

are phospholipase C, phospholipase A2, potassium channels, sodium channels,calcium channels. There are more than 20different G-proteins discovered to date. Afew are listed to the left.

The hormone-receptor mediated processesregulated by G proteins may bestimulatory as in the example of theepinephrine, β-adrenergic receptor, orinhibitory. Each G-protein interacts witha stimulatory G-protein denoted G α s orwith an inhibitory G protein denoted G α i.

Epinephrine also binds to a α -adrenergicreceptor. The α -adrenergic receptor associates with a G α i protein. The binding of epinephrine to the a-adrenergic receptor causes the exchange of GDP for GTP causing the G α i to dissociate from the G βγi

N

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N

NH 2

O

OHO

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HH

O

P

O-

O

N

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N

NH 2

O

OHOH

HHHH

OP-O

O-

O

Phosphodiesterase

N

NN

N

O

O

H 3 C

CH 3

CH 3

Caffeine


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dimer. The inhibition comes from either the G α i subunit associated with adenylate cyclase to directlyinhibit the cyclase, or by the action of G βγi which associates with the G α s subunit when it has GTP bound.The G α i thus competes with adenylate cyclase for G α s.

Cholera ToxinCholera is a gram positive bacterium that causes cholera. Both the cholera bacteria and the toxin remainlocalized in the intestinal epithelial cells. Cholera causes severe diarrhea in its victims which often leadsto death due to severe dehydration. The cholera toxin is protein that catalyzes the ADP-ribosylation ofArg-201 of the G α s subunit we have been talking about. This cholera toxin uses NAD + as a substrate to

produce the ADP-ribosylated Arg-201. The ADP-ribosylation of Arg-201 destroys the GTPase activity ofthe G α s subunit. The G-protein becomes locked into the active state producing prolonged activation ofadenylate cyclase. The elevated levels of cAMP cause the intestinal epithelial cells to secrete highvolumes of fluid.


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The Pertussis Toxin Bordetella pertussis is the bacterium that causes whooping cough. Bordetella pertussis produces a toxinwhich is an enzyme that catalyzes the ADP-ribosylation of a Cysteine residue of the G α i protein. TheADP-ribosylated G α i protein cannot exchange GDP for GTP which prevents G α i from dissociating fromthe G αβγ i heterotrimer. G α i then cannot inhibit adenylate cyclase. Pertussis is a systemic infectionaffecting the regulation of adenylate cyclase throughout the entire body.

Phosphatidyl Inositol Bisphosphate

Cyclic AMP 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 anumber 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 thereceptor induces the associated G protein to exchange GDP for GTP causing the G α q subunit to dissociatefrom the G βγq dimer. The G α q subunit with GTP bound associates with phospholipase C activating thelipase. The activated lipase hydrolyzes the phosphodiester bond linking the phosphorylated inositol to thediacylglycerol. The cleavage of this phosphodiester bond produces 2 second messengers, inositol 1,4,5-triphosphate (IP

3) and diacylglycerol (DAG). DAG diffuses laterally in the lipid membrane while IP

3 is

water soluble and diffuses into the cytosol of the cell.


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Inositol 1,4,5 triphosphate is a second messenger that binds to IP 3-gatedchannels located in the membrane of the endoplasmic reticulum and thecalcisomes. The binding of IP 3 promotes the rapid release of Calcium ions.Calcium ions are yet another second messenger which triggers musclecontractions 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 notopen the calcium channels.

Lithium is used to treat bipolar disorders. Lithium inhibits the recycling ofinositol 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 (PKC). This is a kinase that uses ATP 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 thesequence of the target proteins. This pseudosubstrate sequence occupies the active site of the enzyme

inactivating it. When phosphatidylinositoldiphosphate is hydrolyzed by the activated

phospholipase C, DAG is generated in the lipidmembrane. PKC binds to the DAG creating anattachment of PKC to the membrane. WhenPCK is anchored to the membrane the

psuedosubstrate sequence interacts with head

groups of the phospholipids of the membrane.This activates the kinase activity of thisenzyme. PKC also requires Calcium ions foractivity (remember that IP3 induces the releaseof Ca 2+ from the endoplasmic reticulum and thecalciosomes). The two second messengersDAG and IP3 work in tandem to activate PKC.

DAG also has a short half life. It is phosphorylated to form phosphatidic acid or it is hydrolyzed to itsglycerol and fatty acid components .


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Calcium ions are another important second messenger. The binding of certain hormones and signalmolecules 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 AMPactivates the opening of plasma Ca 2+ channels allowing extracellular calcium to stream in. Within the cellthere are calcium reservoirs such as the endoplasmic reticulum and the calciosomes. The internal storesare not released by cAMP. They only respond to IP3, the second messenger derived from phosphatidylinositol.

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 theannexin proteins. There are over 170 calcium modulated

proteins known. Most of these have the characteristic EFhand. The conformation of these EF hand proteinschanges dramatically when calcium is bound. Shown

below are the conformational changes that occur when calmodulin binds 4 calcium ions. WhenCalmodulin has 4 calcium ions bound, it is bound by target proteins which activate them. One example is

calmodulin dependent protein kinases (CaM kinases). These kinases phosphorylate many proteins thatregulate fuel metabolism, ionic permeability, neurotransmitter synthesis and neurotransmitter release.These kinases are inactive until they bind calmodulin which has 4 calcium ions bound.

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Mechanisms of Signal Transductio1