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Overview of G protein receptor signaling

The G protein coupled receptor mechanism represents the most versatile transmembrane signaling mechanisms.
To see why, take a look at the overview of G protein signaling in the movie to the right .
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(Suggestion: step through the movie, a single step at a time, by repeatedly using the single arrow to right button; review the uninterupted movie using the double arrow to right  button)

Ligand binding in a GPCR

Given to the left is a movie clip showing how noradrenaline might bind to the receptor in the ligand binding pocket of a GPCR .

The pocket is formed from fransmembrane regions III, IV, V and VI.

Amino acids (with position numbers) important to noradrenaline binding in the site are indicated.

Imagine that you are in the ligand binding pocket, looking out to the extracellular space.

You see a noradrenaline molecule in the distance which is homing in on the receptor binding site.

Notice how the noradrenaline makes a perfect fit in the binding site to form (1) an ion pair, (2) two hydrogen bonds and (3) an  hydrophobic interaction with the hydrophobic ring of phenylalanine.

The binding of noradrenaline to the receptor will induce conformational changes in the receptor leading to receptor activation.

These types of models have proven very valuable in helping design new receptor agonists and antagonists for G protein linked receptors.

Activation of a GPCR

The principle of receptor signaling through G proteins is that the ligand (e.g. a neurotransmitter or neuropeptide) induces a change in the sturcture of the receptor such that the receptor-ligand complex can activate the G protein (see movie to the right).

Intracellular loops of the receptors are responsible for the activation of the G proteins.

The G proteins associated with receptor transduction are the so-called "trimeric" G proteins because they are composed of three subunits, alpha, beta and gamma.

The trimeric designation distinguishes the receptor-associated G proteins from smaller intracellular "monomeric" G proteins that are involved in vesicular traffic and other processes within the cell.

After activating a G protein the ligand-reecptor complex remains active and can activate more G proteins.

In this sense, one can thing of the activated ligand-receptor complex as an enzyme.

Activation of a G protein

This animation showing a GTP replacing a GDP during activation of a G protein.

Does it look like the GTP is having trouble kicking the GDP out of the binding site in the alpha subunit? This is because this replacement is the rate limiting step in the GTPase cycle (this is the step which receptor-ligands catalyze).

Now let's compare the basal activity of a G protein (left) with that of a G protein that is associated with an activated receptor-ligand complex (right).  

This animation requires a little patience as in each case it goes through four GTP hydrolysis steps. Note how much faster it is in the presence of an activated receptor-ligand complex.

The action of cholera toxin on Gs proteins

Cholera toxin can block Gs proteins in their active form. The flash animation to the right illustrates how this occurs. The toxin penetrates the membrane to block the G protein.

source: Tutorials in Cell Signaling

The inhibition of Gi proteins bypertussis toxin

Pertussis toxin blocks Gi proteins in their inactive form. A subunit of the toxin penetrates the membrane and must be activated intracellularly before it work.

The action of a kinase

Kinases are enymes that catalyze the phosphorylation of proteins on the hydroxyl side chain group of the amino acids serine, threonine or tyrosine. This animation gives an overview of the events occuring in the kinase during a phosphorylation.

The activation of Protein Kinase G (PKG) by cyclic GMP

PKG is a dimer, composed of two regulatory domains (shown in blue/purple) and two catalytic domains (green).

The catalytic domain and the regulatory domain are contected by a hinge domain (black) in each subunit.

The dimer has four cGMP binding sites and two kinase domains.

To activate the kinase four cGMP molecules must bind to the nucleotide binding sites.

Notice how the pseudo-substrate masks the kinase site in the inactive kinase.

Binding of cGMP causes the kinase to flip open by the hinge thus exposing the kinase site to an authentic substrate.

The substrate can then bind to this site and is subsequently phosphorylated with the gamma phosphate group of ATP acting as the phosphate donor( see previous movie) (note: ATP binding site is indicated in orange; ATP is in red).

The phosphorylated substrate  then leaves the kinase, ADP is replaced by ATP, and the kinase is ready to phosphorylate another substrate.

The kinase returns to the inactive form when cGMP leaves the nucleotide binding sites.

The activation of Protein Kinase A (PKA) by cyclic AMP

In PKA the regulatory and catalytic parts of the kinase are two seperate proteins rather than two domains connected by a hinge domain.

The enzyme is composed of two catalytic subunits (indicated in green in illustration) and two regulatory subunits (indicated in blue/purple).

The two regulatory subunits are held together by a so-called leucine zipper (dark purple).

Cyclic AMP binds to the regulatory subunits.

Upon activation by cylic AMP the complex breaks apart  and the catalytic subunits are free to diffuse throughout the cytoplasm.

It can even diffuse into the nucleus.

In the inactive kinase "pseudo-substrate" domains of the regulatory subunits bind to the catalytic sites (dark green) of the catalytic subunits.

The activation of Protein Kinase C (PKC)

PKC has a hinge domain, similar to PKG.

PKC lacks the leucine zipper in the regulatory subunit and thus it does not form a dimer.

For activation PKC requires diacyl glycerol (DAG), phosphatidyl serine (PtdSer) and Ca2+.

There is a site for each of these products in the regulatory domain of PKC.

The name PKC comes from Ca2+ because it was first thought that Ca2+ was the critical factor activating this kinase.

It was subsequently shown that only low levels of Ca2+ are required to meet the Ca2+ requirements of this kinase. 

Indeed, basal (resting) levels of Ca2+ are often sufficient, and thus it is the presence or absence of DAG which has proved to be the more important dynamic regulator of this kinase. 

DAG is very hydrophobic and, following its synthesis, stays in the membrane.

Thus, not surprising, PKC usually functions near the membrane.

In fact, DAG is though to draw the kinase into a close association with the membrane, as illustrated in the movie clip.

There is also a membrane bound protein called "Receptor for Activated C Kinase" (RACK), which is thought to be important in anchoring the activated kinase in the membrane.

Notice in the animation that while both phosphatidyl serine (PtdSer, a phospholipid) ) and Ca2+ (green ball) are necessary for kinase activity, it is DAG that is determining kinase activity.

The PtdSer and Ca2+ are normally present at sufficient levels to support enzyme activity.

This is not true for DAG, and thus it is the most important factor in determining PKC activity.

The structure of Calcium Calmodulin-dependent Protein Kinase (CaMKII)

Given to the left is the structure of CaMKII.

Its structure is similar to PKG, PKA and PKC but the regulatory domain is located in the C-terminal region and the catalytic domain in the N-terminal region.

Like the other kinases it possesses an ATP binding site near the kinase domain.

It also possesses a hinge domain.

With folding the so-called inhibitory domain or pseudo-substrate domain comes to rest in the vicinity of the kinase domain, thus ensuring that its activity is blocked.

CaMKII also has a phosphorylation site which, as you will see in a series of movies below, is very important to the functioning of this kinase.

Play the movie to see how the folding can bring the various functional domains of the kinase in the correct orientation.

The activation and inactivation of CaMKII

To the right and below are a series of movies  showing how Ca2+-calmodulin dependent protein kinase II works.

The first movie shows activation under conditions where there is only a very short duration increase in Ca2+.

Under the conditions of a short duration increase in intracellular Ca2+ the CaMKII activity is, and remains, directly dependent on Ca2+-calmodulin.

In other words, if there is no Ca2+-calmodulin present, the kinase is inactive.

When the Ca2+ returns to low basal levels the ions quickly dissociate from the calmodulin and the protein dissociates from the kinase.

The kinase then quickly returns to the closed, inactive, state.

The movie to the left shows what happens to CaMKII when the Ca2+ level remains high for a longer time.

In this case the enzyme is in the open state sufficiently long to be phosphorylated by another CaMKII.

The kinase cannot phosphorylate itself, thus this is not an autophosphorylation but a transphosphorylation.

In the movie note how the phosphorylated CaMKII tries to return to the inactive state but can't due to the phosphorylation.

This is the reason this kinase has been referred to as the kinase with a memory i.e. it remembers having been activated by Ca2+-calmodulin, and thus remains activated.

This memory is important to the function of CaMKII, particulaly in the brain.

The enzyme becomes inactivated only after an enzyme called a phosphatase removes the phosphorylation of CaMKII.

Construction of a integrating machine with CaMKII

In considering how phosphorylation of CaMKII can be a useful mechanism, consider what would happen if two CAMKII molecules were complexed or tied together.

The chances or probability of one CaMKII finding the other, for transmolecular phosphorylation, would be drastically increased in comparision to completely free enzymes.

If three kinase molecules were in the complex then the probability of transmolecular phosphorylation is increased even further.

In fact, CaMKII often exists in a complex considing two rings, each ring containing six CaMKII molecules.

Thus the complex contains a total of 12 kinase molecules, i.e. it is a dodecameric holoenzyme.
The animation to the left gives an impression of the structure of such a complex.

The complex is held together via so-called "self-association" domains, which are found towards the C-terminal of the kinases, in a region which contains variable inserts.

Only isoforms of the enzyme which contain the association domains participate in forming the complexes.
If a cell makes such isoforms depends on how it alternatively splices the primary mRNA transcript during gene expression of the CaMKII gene.

Splicing in appropriate inserts will lead to the construction of  a dodecameric CaMKII holoenzyme.

The self-associaton domains form a narrow stalk to the central mass of the complex.

This complex can become an integrating machine because as more enzymes become phosphorylated (and thus permanently activated) the probalbility of a newly activated enzyme itself becoming phosphoylated increases.

This is because the probability that its neighbour is active has increased.

This complex can be used to detect changes in the frequency of Ca2+ signaling (if frequency is high then the CaMKII is partially activated by the time the next Ca2+ signal comes).

If the Ca2+ signal is strong enough then all enzymes of the holoenzyme become phosphorylated and thus remain active after Ca2+ returns to basal levels.

Given below are three movies showing how the holoenzyme complex function under conditions where a low, medium or high Ca2+ signal has been introduced inside the cell.

The figure on the right gives how the various components of the CaMKII holoenzyme signaling system are represented in the movies.

Run each of the movies and then compare the situation at the end of each movie (where the Ca2+ signal has dissappeared) with respect to the degree of activity of the holoenzyme.

Movie 1: low Ca2+ signal

Movie 2: medium Ca2+ signal

Movie 3: high Ca2+ signal

Calcium Wave

Calcium waves can be generated in cell through mobilization of intracellualr Ca2+ stores. There are two way to initiate such waves, either through an influx of Ca2+ from the extracellular space through e.g. voltage-gated ion channels or through the production of IP3. The Ca2+ or the IP3 act on IP3 receptors on the ER to initiate the Ca2+ wave which then becomes self-propagating through the process of Ca2+ induced Ca2+ release (CICR). At high Ca2+ concentrations the IP3 receptors are inhibhted (they close) thus terminating the Ca2+ signal. Ca2+ waves can be sent throughout the cell with this CICR mechanism, and in some cases the the Ca2+ wave even enters the nucleus.

Given below are two movies, one showing the Ca2+ wave being initiated by an influx of Ca2+ and the other through production of IP3

Wave intiated through Ca2+

wave initiated through IP3 (the IP3 is represented by the blue balls).