BIO254:2ndMessenger

=Introduction= Secondary messengers are a class of small molecules used to transduce extracellular signals to the interior of the cell. Most hormones such as epinephrine and seratonin are too large or too polar to freely pass through the plasma membrane of a cell. For example, second messengers are often free to diffuse to the nucleus, where they can influence gene expression and other processes. The use of a membrane-bound protein with intra- and extracellular domains allows information to pass through the membrane without any exchange of contents. In addition to transducing a signal through the membrane, the use of secondary messengers allows for signal amplification since one bound primary messenger can stimulate the production of many secondary messenger molecules. For instance, enzymes or membrane channels are almost always activated in second-messenger generation; each activated macromolecule can lead to the generation of many second messengers within the cell. Thus, a low concentration of signal in the environment, even as little as a single molecule, can yield a large intracellular signal and response. Secondary messengers can be either small organic molecules such as cyclic adensine monophosphate (cAMP), guanosine 3',5'-(cyclic)phosphate (cGMP), diacylglycerol (DAG) and inositol 1,4,5 triphosphate (IP3) or inorganic ions such as Ca2+ (Stryer, 2005).



=Major secondary messenger=

The Epinephrine Cascade
Many hormones use cAMP as a secondary messenger. The first such system investigated was the breakdown of glucose triggered by epinephrine. Since then, cAMP has been discovered to play a role in the action of vasopressin, liptropin, glucagon, calcitonin, thyroid-stimulating hormone and many other primary messengers. This large number of pathways utilizing a common messenger increases the potentiona for "cross talk" between signaling events. Recent work has indicated the importance of both spatial and temporal control of cAMP concentration. The exact nature of these controls is still an active area of research (DiPilato et al, 2004).

Activation
Once epinephrine is released and travels to the target cell surface, it binds to the extracellular domain of the seven helix transmembrane protein β-andrenergic receptor. The cystolic domain then participates in activation of Gs, a G protein. The activated Gs then releases its α domain along with a bound GTP. This Gα-GTP in turn stimulates activity of adenylyl cyclase (AC), which catalyzes the transformation of ATP into cAMP. AC is primarily bound to the cell membrane, and it is this form that is responsible for transmitting the epinephrine cascade. The other form of AC is free in solution and localized in the nucleus, mitochondria and microtubules. Soluble AC is activated primarly by bicarbonate and calcium (Nahirney et al, 2003).





Actuation
The secondary messenger cAMP eventually triggers the phosphorylation of a number of other proteins, but this not accomplished by cAMP itself. Instead, cAMP activates a protein kinase, usually protein kinase A (PKA). Binding of cAMP releases the regulatory domains of PKA and allows the catalytic domains to transfer a phosphoryl group from adenosine triphospate (ATP) onto the target molecule. This reversible phosphorylation directly changes the activity or function of the affected molecule.

Click here for a 3D rotating view of PKA showing the two regulatory domains (gray and tan) and the catalytic domain (blue) (Linked from the Lawrence Berkeley Advanced Light Source http://www-als.lbl.gov/als/)

Regulation
Deactivation of the cAMP cascade produced by epinephrine activity is a two step process. First, Gs acts as a GTPase, hydrolyzing GTP in the Gα-GTP complex in order to deactivate it and prevent further activity of AC. In the second step, β-andrenergic receptor kinase phosphorylates the cystolic tail of the β-andrenergic receptor to deactivate it. β-arrestin then caps the phosphorylated tail. In addition to these measures, which prevent production of new cAMP, the enzyme cAMP phosphodiesterase also contributes to regulation by degrading existing cAMP.

cGMP
Cyclic GMP is very similar to cyclic AMP. It was discovered in urine in 1963 and considered a second messenger through investigating the mechanism of transducing the visual signal from photobleached rhodopsin to the cationic channels in the plasma membrane in the 1970's. The regulation of its synthesis has remained a mystery until recently.

Transducin, a G protein, which is made up of three different subunits: Tα, which is the guanyl nucleotide binding subunit, T β, and Tγ. In the dark, with GDP bound, transducin is peripherally associated to the cytoplasmic disc surface. In the first step of the light triggered cascade, photoactivated rhodopsin (R*), the photoisomerized protein, forms a transient complex with transducin and catalyzes on it a GDP/GTP exchange. GTP-bound transducin binds to and activates a cGMP phosphodiesterase (PDE), which converts cGMP to GMP. Cyclic GMP is formed from GTP by guanylate cyclase and is hydrolyzed to 5’-GMP by the phosphodiesterase as Figure 4. The concentration of cGMP decreases below what is required to open cGMP-gated ion channels of the rod, reducing the flow of ions across the cellular membrane. (Fung, B. K. et al, 1981)



Transducin can returns to the inactive state by hydrolysis. It bound GTP molecule into guanosine diphosphate, GDP. Because transducin bound to GDP has a low affinity for PDE, it releases the enzyme in an inactive form, allowing cGMP levels to rise again and return the flow of ions across the cell membrane to the 'dark' state. Also, photoreceptor cells possess a protein called regulator of G-protein signalling 9 that accelerates transducin's ability to hydrolyse GTP. Photoexcited rhodopsin is deactivated by phosphorylation and then capped by arrestin. (He, W. et al, 1998)



Inositol trisphosphate (IP3) and diacylglycerol (DAG)
A wide variety of G protein-coupled receptors link to phospholipase C (PLC), intracellular enzyme, to hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2)generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These two second messengers in turn regulate intracellular Ca2+ concentration and protein kinase C activity.

Inositol-1,4,5-trisphosphate (IP3)
IP3 is a second messenger for many growth factors, hormones, and neurotransmitters. This soluble molecule diffuses through the cytosol and binds to IP3 receptors on the endoplasmic reticulum and nuclear membrane causing the release of calcium from storage vesicles into the cytosol. These signals are terminated by the dephosphorylation of IP3 or converting into derivatives.

Diacylglycerol (DAG)
DAG remains in the inner layer of the plasma membrane. It recruits Protein Kinase C (PKC), a calcium-dependent kinase, which phosphorylates many other proteins that bring about the changes in the cell. Activation of PKC requires calcium ions. These mechanisms are collaborated with IP3. These signals are terminated by phosphorylating DAG to arachidonic acid or hydrolyzing DAG to glycerol or fatty acids.



Calcium ions
Calcium ions are probably the most widely used intracellular messengers. In response to many different signals, a rise of the Ca2+ concentration in the cytosol triggers many types of events, such as secretion, contraction, gene expression and apoptosis development. Normally, the level of calcium in the cell is very low (~100 nM). However, its level in the cell can rise dramatically. There are two major types of pathways to sense the concentration change of calcium inside the cell.


 * Binding of Ca2+, calmodulin is activated when the cytosolic calcium level is raised. Calmodulin is a member of the EF-hand protein family. The EF hand is a Ca2+-binding motif that consists of a helix, a loop, and a second helix, originally discovered in the protein parvalbumin. Calmodulin protein structure changes depending on binding Ca2+ or not. Calmodulin- Ca2+ complex stimulates a lot of targets such as CaMKII, which can phosphorylate multiple downstream enzymes and proteins to induce various responses.


 * The other example is synaptotagmin which serves as the major Ca2+ sensor for regulating exocytosis from neurons. Ca2+ triggers two consecutive synaptotagmins interact with each other, via coupling its second C2 domain.


 * To get more examples of Ca2+ regulating pathways, you can reference: ACTIVITY-DEPENDENT TRANSCRIPTION.

=References= 1. Stryer, L. Biochemistry 4th Edition. New York. WH Freeman And Company, 1995

2. Lisa M. DiPilato, Xiaodong Cheng, and Jin Zhang, "Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments" PNAS (2004) 101, no. 47, 16513-16518

3. Nahirney, PC; Zippin, JH; Kamenetsky, R; Chen, YQ; Levin, LR; Buck, J; Fischman, DA "Cell and tissue localization of bicarbonate sensitive adenylyl cyclase" FASEB JOURNAL, 2003, 17, no.5, pt.2, suppl.S, p.A785-A785

4. Fung, B. K.-K., Hurley, J. B., Stryer, L. Proc. Natl. Acad. Sci.(1981) 78, 152-56

5. He, W., Cowan, C. W. & Wensel, T. G. Neuron, (1998) 20, 95−102

6. Tod R. C., Leslie M. S., Robert F. M. and Sue C. K., BMC Neuroscience (2001) 2, 6

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