Gs, The different isoforms of G-proteins mentioned above,

Gs, Gi and Gq are present in a family
of proteins called heterotrimeric G-proteins. These proteins are formed by a
complex of three different parts: alpha, beta and gamma subunits. Additionally,
they are named ‘G’ proteins as they bind to purine nucleotides of guanine
called guanine triphosphate (GTP) and guanine diphosphate (GDP). G-proteins are
considered a class of membrane-bound proteins that regulate protein function,
leading to control over the concentration of second messengers through a
signalling cascade. There are four different isoforms that G-proteins can be
divided into: Gs, Gi, Gq and G12/13; however, this essay will
explore the signalling pathways downstream of Gs, Gi and Gq. The signalling
pathways involve G-protein coupled receptors (GPCRs) and secondary signalling
cascades which regulate the function of specific proteins. Understanding of the
function of these pathways begins with the roles of the various subunits.

 

The heterotrimeric G-proteins all
contain the three parts, with alpha being the largest and most active subunit.
They are specialised proteins with the ability to bind guanine nucleotides. The
alpha subunit is considered the most active as it is the binding site of GTP
and GDP which regulate the secondary cascade but the beta-gamma complex can
mediate the same amount of functions. The GTP or GDP is often considered
another subunit of the G protein as it regulates the function of the protein
with its target. G proteins are very diverse as some can stimulate activity at
their target proteins and others inhibit it. The G protein is associated to the
plasma membrane at the alpha and gamma subunit by lipid anchors. The beta-gamma
subunits always remain as a heterodimer but can regulate function as a complex
as well as modulating the alpha subunit. Each subunit is a protein composed of
different amino acids therefore they have different structures.

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Generally, G-proteins function can be
described through the interaction of the different subunits with the target
proteins. As G-proteins are linked with the inner surface of the plasma
membrane, they interact with various intracellular proteins such as kinases,
channels and GPCRs. They perform a signalling cascade by acting as signal
transducers to communicate between extracellular hormones and neurotransmitters
and their intracellular targets. The GPCRs are the link between the
extracellular ligand, and the G-protein which will regulate intracellular
function. The different isoforms of G-proteins mentioned above, have different
effects on the proteins they regulate which leads to differences in
concentrations of the second messengers. GPCRs form receptors from many
different functions in the body, including adrenergic amines, acetylcholine and
visual receptors to name a few.

 

Approximately 865 genes in the human
body code for GPCRs as they are so common and variable but only found in
eukaryotes. Humans have over 1000 known GPCRs and each one has a specific
function. They are activated by agonist ligands that cause a conformational
change which is transmitted through the seven cytoplasmic, transmembrane loops
of the receptor. The seven transmembrane alpha helices give it an unique
appearance, specific to its function and secondary cascade. The conformational
change leads to activation of the alpha site of the G-protein through binding
of GTP in replacement of GDP. The G proteins transduce signals from many GPCRs
to the effector proteins such as ion channels and enzymes. Following this,
changes in intracellular production, secretion or reduction of secondary
messengers occurs through regulation.

GPCRs function under a lock and key
mechanism with one or a few specific protein molecules. When the complementary
protein ligand fits into the GPCR, it will result in a conformational change of
the GPCR which triggers a complex secondary cascade, resulting in specific cell
functions. As the conformational change occurs, the alpha subunit of the G
protein exchanges the GDP for GTP. As GTP is bound to the alpha subunit, it
will dissociate from the beta-gamma complex and bind to its target protein to
regulate it. The alpha subunit and beta-gamma dimer can both interact and
regulate the function of specific target proteins. Once activated, the target
protein can relay signals via second messengers. This process between the GPCR
and the G protein in the membrane can occur continuously while the
complimentary ligand is bound to the GPCR site. The last process to
re-establish the resting state is achieved through the hydrolysis of GTP to
GDP. As GDP is now bound to the alpha subunit, it re-associates with the
beta-gamma dimer and the ligand detaches from the GPCR. The regulation of the
process back to the normal state, has many methods in the body. The main way is
through the RGS (regulation of G protein signalling) protein.

Water soluble hormones are an example
of ligands that can bind to transmembrane proteins such as GPCRs. They are
hydrophilic, and therefore cannot diffuse through the lipid bilayer. A
technique for their action is binding to protruding integral transmembrane
proteins in the plasma membrane. The hormone binding to the receptor is a first
messenger action, which leads to secondary messenger activation. The binding
leads to G-protein molecules being activated which leads to a secondary cascade
of events. An example of this binding is the G protein activation in skeletal
muscle. The action of epinephrine (adrenaline) on skeletal muscle highlights
the role of G-proteins in the regulation of cyclic AMP (cAMP). As skeletal
muscle stores glucose as glycogen, when exercising, ATP is used in muscle
contraction to convert the glycogen back to glucose. This process is initiated
by epinephrine secreted into the bloodstream from the adrenal medulla.
Increased epinephrine results in activation of the beta adrenoreceptor on the
muscle membrane. This receptor is linked to G-proteins, as it stimulates the
dissociation of the alpha subunit from the beta-gamma dimer and activates
adenyly cyclase. The next step is the increase in cAMP which leads to a further
chain of enzyme activation which will eventually lead to the breakdown of
glycogen in glucose.

 

Gs and Gi proteins have opposite
functions on the concentration of cyclic AMP (cAMP). Receptors using Gs protein
class are beta 1, beta 2, dopamine 1, histamine 2 and vasopressin 2. When these
receptors are stimulated, it causes the Gs protein to activate adenlyl cyclase
which converts ATP into cAMP. cAMP activates protein kinase A which increases
intracellular levels of calcium in the heart, leading to heart muscle
contraction, smooth muscle relaxation and glycogenolysis. On the other hand,
receptors using Gi class are M2, alpha 2 and dopamine 2. When these receptors
are stimulated, the Gi protein will inhibit adenyly cyclase which leads to a
decrease in cAMP and therefore a decrease in protein kinase A. The Gi protein
function example is when somatostatin inhibits the release of gastrin by G
cells in the gastric mucosa. This is the main signalling cascade through
activation of these G proteins. The cAMP formed due to receptor activation
binds to other proteins such as enzymes and ion channels to regulate their
function. The change in concentration of this molecule regulates the function
of other proteins through the Gs and Gi proteins. Furthermore, once the adenyly
cylase is activated, it can produce many molecules of cAMP, so the cell can
amplify the initial signal many times. Only one cAMP molecule is needed to
activate another protein in the cell but the signal can be terminated through
the conversion of cAMP to AMP by phosphodiesterases.

 

The final form of G proteins discussed
are Gq proteins. Receptors using G protein class Gq are H1, alpha 1, V1, M1 and
M3. This is an important second messenger cascade starting at phospholipids in
the inner lining of the plasma membrane. The phospholipid commonly described is
phosphatidyl inositol 4,5-bisphosphate. When the acetylcholine acts as the
binding ligand, muscarinic G protein receptors activate an enzyme called
phospholipase C, the plasma membrane cleaves the phospholipid into PIP2. The
PIP2 then splits by hydrolysis into two molecules: diacylglycerol (DAG) and
inositol 1,4,5-triphosphate (IP3). Both products act as
intracellular mediators and initiate a secondary messenger cascade. IP3 is a
water-soluble molecule that binds to an IP3 receptor to mobilise calcium from
the store in the endoplasmic reticulum to be released intracellularly. The
increase in calcium leads to activation of calcium depend events such as smooth
muscle contraction and enzyme secretion by pancreatic acinar cells. DAG is a
hydrophobic molecule that is therefore retained in the membrane. It can diffuse
in the plane of the membrane to stimulate activation of protein kinase C.
Protein kinase C activation leads to phosphorylation of target proteins and
regulation of various physiological responses. This is the secondary messenger
cascade for the Gq protein.

 

Certain G proteins can be permanently
activated by bacterial toxins. The change to the protein occurs through
covalent modification of the alpha subunit of the G proteins or through
mutations in the generation process of cyclic AMP. An example of this is cholera
toxin in intestinal epithelial cells. It is a deadly toxin produced by cholera
bacteria. It modifies the G proteins in intestinal epithelial cells so they
become permanently activated. This leads to a huge increase in the
intracellular cAMP concentration. A secondary process of cAMP in these cells is
to stimulate active transport of Cl- ions out into the lumen of the intestines.
Due to a build up of Cl- ions in the lumen, water flows out of the cells via
osmosis and Na+ ions also follow the Cl- ions as they are attracted to the
negative charge build up in the lumen. Cholera toxin effectively results in
sodium and chloride ions and water being excessively ejected into the faeces.
The treatment for this involves a replacement of the lost fluids through oral
rehydration therapy or intravenously and antibiotic therapy with tetracycline.
Although these G-proteins are permanently activated, there are methods of
treatment to stop their effect and remove the initial binding ligand.

In conclusion, the heterotrimeric
G-proteins can be divided into three forms: Gs, Gi and Gq. They each have a
distinct signalling pathway which occurs due to the change in the subunits:
alpha, beta and gamma. The alpha subunit dissociation due to a change from GDP
to GTP results in a secondary cascade of events which regulate the
intracellular concentrations of various molecules. Gs and Gi affect cAMP
concentrations and Gq affects DAG and IP3. The G proteins are linked to GPCRs
which are activated when a ligand binds to their initial extracellular binding
site via a lock and key mechanism. These receptors are unique as they have
seven transmembrane alpha helices. Finally, certain G proteins can be activated
in a permanent state due to bacterial toxins. This occurs as the binding to the
GPCR is constant which stimulates the activation of the G protein and the
following secondary cascade of events of the different G proteins.

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