Molecular Pharmacology - anno 2023/2024 completo

SIGNAL TRANSUCTION.

LGCI transform a chemical signal into an electrical one. This kind of signal is very fast. It takes milliseconds to

be released and to be transmitted. If we compared the timing needed to have the signal transduction of this

receptor to the other classes of receptor, we have a score in timing. The G protein coupled receptor are able

to activate a signal transduction that require seconds. The enzyme receptors (TRK) will require minutes. And

the DNA/RNA receptors (gene transcription) require hours.

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5.2 T G-

HE COUPLED PROTEIN RECEPTORS

A variety of hormones, neurotransmitters, and other chemical mediators induce their responses by coupling

to G proteins, which in turn regulate specific effectors. G‐protein‐coupled receptors (GPCRs) represent the

largest family (800 genes) of membrane receptors. GPCRs recognize ligands as different as catecholamines,

serotonin, acetylcholine, GABA, glutamate, glycoprotein hormones, peptides, and molecules involved in cell

adhesion.

✓ Hormones

✓ Neurotransmitters

✓ Light sensitive compounds

Each receptor is formed by a single polypeptide chain that spans the plasma membrane seven times (seven

transmembrane receptors: 7TM) with extracellular N‐terminus and intracellular C‐terminus.

They need to be coupled to a G-protein, formed by three different subunits (α, β, γ). The alpha subunit is

specific (it gives specificity).

The alpha and gamma subunit of the receptor are bound to the plasma membrane. The beta subunit is not

anchored to the membrane and can move inside the cytoplasm. Alpha subunit binds either GTP or GDP

depending on whether the protein is active (GTP) or inactive (GDP).

Some G proteins, such as the signaling protein Ras, are small proteins with a single subunit.

How they work?

When the hormone or neurotransmitter receptor is not activated by its

specific agonist, the guanine nucleotide binding site in the α‐subunit is

occupied by GDP and α remains tightly associated with the βγ complex as an

inactive heterodimer. Upon ligand binding, the activated receptor interacts

with the G protein and induces GDP dissociation and subsequent GTP binding

to the α‐subunit. This event causes a conformational change in the α‐subunit

resulting in its dissociation from the βγ complex. Then, both α‐GTP and free

βγ complex interact with downstream effectors regulating their activity.

The signal is terminated when the GTPase activity of the α‐subunit hydrolyzes

GTP to GDP and α‐GDP associates with the βγ complex, forming again an

inactive heterotrimer. To ensure a rapid inactivation of G‐protein signals, GTP

hydrolysis is regulated by proteins capable of interacting with the α‐subunits.

GPCRs are involved in information transfer (signal transduction) from outside

nd

the cell to the cellular interior. They produce 2 messengers.

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LESSON 9 – 31/10/2023

5.2.1 Three main effector pathways are regulated by this class of receptors:

A single receptor can regulate more than one effector, thus initiating a complex network of coordinated

signaling events by the interaction of both α‐GTP and the βγ‐subunits.

It is believed that different active conformations of receptors mediate binding to different G proteins. Certain

agonists cause a receptor to preferentially interact with a specific G protein.

I. ACTIVATION OF ADENYLYL CYCLASE PATHWAY.

It’s a membrane-associated enzyme (2 transmembrane regions and 2 cytoplasmatic regions). When

it’s activated by the GTP bound to a type of alpha subunit, it catalyses synthesis of the second

messenger cAMP from molecules of ATP, or inhibits it (α -GTP stimulates and α -GTP inhibits). Some

s i

receptors stimulate adenylyl cyclase activity, whereas others inhibit it.

There are 9 main forms of adenylyl cyclase. All these enzymes share a similar structure consisting of

a short N‐terminal segment and two large and highly homologous cytoplasmic domains (named C1

and C2) separated by two highly hydrophobic regions (M1 and M2) that span the plasma membrane

six times.

In humans, cAMP is involved in responses to

sensory input, hormones, and nerve

transmission, among others.

Then cAMP molecules (when increase at

intracellular level) bind the PKA (protein kinase

A) and cause the release of catalytic subunits,

that phosphorylate a variety of proteins. PKA is

normally composed of 2 regulatory subunits and

2 catalytic subunits. PKA activation by cAMP is

involved in the control of multiple cell functions

including ion channel activity, metabolic

reactions, gene transcription, cardiac activity,

smooth muscle cell relaxation, glycogenolysis

and lipolysis, secretion, differentiation, and proliferation.

An example could be the troponin protein that is phosphorylated and activated by the PKA. There

are 3 types of troponin: the troponin C binds calcium (increases the interaction with calcium, the

troponin T binds the tropomyosin and allows the muscle contraction and the troponin I is the

inhibition subunit. The PKA can also phosphorylate the phospholamban (PLN), a small and reversible

2+

transmembrane protein located in the cardiac sarcoplasmic reticulum. It regulates the activity of Ca

pump and the relaxation of the muscle, that is when PLN is phosphorylated it actives the calcium

pump and the muscle relaxation is faster. It removes from the cytoplasm, accumulates it and releases

it for another contraction.

cAMP is hydrolyzed by phosphodiesterase (PDE).

II. ACTIVATION OF PHOSPHOLIPASE C PATHWAY.

Phospholipase C. a phosphodiesterase, hydrolyzes phosphatidylinositol 4,5‐ bisphosphate (PIP ) and

2

produces two second messengers, IP and DAG, which regulate many cell functions such as

3

metabolism, secretion (modulation of neurotransmitters and hormone release), muscle cell

contraction, neuronal activity, inflammation, ion transports, proliferation/tumor formation MAPK,

migration, and proliferation. The diacylglycerol is very nonpolar (it diffuses in membrane) and

activates protein kinase C (PKC) and inositol 1,4,5‐trisphosphate (Ins‐1,4,5‐P3) is polar in the

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2+

cytoplasm interacts with its specific receptor (R) to release Ca from intracellular stores. DAG is

rapidly metabolized by a specific lipase or by a kinase that converts it into phosphatidic acid. IP3 is

rapidly metabolized in the cytoplasm by two enzymes (phosphatase and kinase).

Lithium, a drug used for the treatment of bipolar disorder, inhibits inositol monophosphatase, one

of the phosphatases involved in inositol phosphate metabolism, thus causing inositol 1‐phosphate

accumulation and interruption of phospholipid resynthesis cycle. This inhibition modifies neuronal

activity.

For instance, thrombin receptors in platelets use this pathway to promote blood clotting.

III. Interaction of GPCRs with OTHER PROTEINS.

Activation of Ion channels mechanism, triggering flow of ions and induce changes in cell excitability.

An example is metabotropic glutamate receptor. A metabotropic receptor is a receptor that binds

the ligand and it transmitters the signal by several reactions that form a cascade. A subtype of

membrane receptors that do not form an ion channel pore but use signal transduction mechanisms,

often G proteins, to activate a series of intracellular events using second messenger chemicals.

GPCR classification:

G PROTEIN RECEPTOR SIGNALING PATHAWAY

G Associated with beta Increase adenylyl cyclase Activate cAMP

S adrenergic receptors, cAMP and excitatory Calcium channel

glucagon, histamine, effect

serotonin

G Alpha2 adrenergic Decrease cAMP Inhibits cAMP

i receptors, mAchR, Cardiac K+ channel open Activates K+ channel

opioid, serotonin Decrease heart rate

G Coupled to mAchR, PLC-IP3, DAG Activate PLC

q serotonin, 5HT Increase cytoplasmatic

1C Ca2+

G Rhodopsin and colour Increase cGMP Activate cycling cGMP in

t opsin in retinal rod and phosphodiesterase the eyes

cone cells Decrease cGMP

Videos of action and desensitization of these receptors:

http://www.youtube.com/watch?v=gPyo7k9E_-w

https://www.youtube.com/watch?v=IP4zsbemW8I 13 Bacci Anna – University of Verona

To prove the existence of different types of GPCR in the 1970’s they developed the technique of radioligand binding. Radioligand

binding is an approach that makes use of a radioactively labeled compound, which binds at the target binding site. This permitted

the direct measurement of receptors (regulation, action, subtypes and drug discovery).

They isolated the receptors with affinity chromatography (high purification).

They prove the receptors exist as specific isolated molecules.

In 1980s they did the receptor reconstitution (take the receptors purified and reconstituted them in the phospholipid vesicles). They

put these vesicles in a cell previously deprived of these receptors. There is the fusion between cell membrane and vesicles. The cells

become responsive to the ligand of these receptors added. This is a proof of receptor specificity (because without these receptors

the cell in the presence of ligand do not respond to it).

Rhodopsin discovered by classical Edmond degradation, so protein sequencing (not by cloning) because there are so much of this

proteins.

➔ To sum up: a large family with different assignment.

Chimeric receptors and receptors mutagenesis that change the specificity of G-protein coupling (depending on the residues near the

cell membrane). With these mutations they believed to find a loss of function of the receptors, but they found out that the receptors

led to generation of second messengers even in the absence of the ligand. They called that constitutively active mutant receptors.

This happened because by modifying the receptor (with mutagenesis) they change the affinity of receptors that became constitutively

active because more molecules can interact with the receptor.

They found out that lots of disease are caused by mutations of heptahelical receptors.

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Desensitization when a cell is stimulated with any agonist, the results of that stimulus wane. The cell wants to return at original

state. By using the example of beta-adrenergic receptor, when it is stimulated it leads through the G-protein arm to activation of

second messenger signalling. But the active receptor is recognized by a kinase called GRK2. It phosphorylates the activated form of

the receptor and facilitates the binding interaction of beta-arrestin. This one sterically blocks interaction of the G-protein, thus

leading to a waning of stimulation, and desensitization of the receptor. But also the classical feedback regulatory loops whereby an

enzyme activated, through the G-protein, can feedback regulate the receptors by phosphorylating it.

A desensitized receptor is retarded in a SDS polyacrylamide gel as compared to normal receptor because of the modifications of

proteins.

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