Week 3 (topic 3)

Question 1

What are the properties of ligands defined by?

Answer 1

Their action at the receptor site

Question 2

What are the key features of agonists?

Answer 2

Agonists have affinity:
→ The tendency to bind to a receptor
Agonists have efficacy:
→ The tendency to elicit a response when bound = efficacy

Question 3

What is the law of mass action?

Answer 3

Describing the affinity of agonist and antagonist binding to receptors
This very useful property: allows us to quantify the agonist-receptor effects + antagonist potency

Question 4

What are the 2 factors that affect the function/ responsiveness?

Answer 4

a) receptor occupation by agonist (affinity)
b) agonist action at receptors (efficacy)

Question 5

What are the 2 main reasons why adding an agonist will only produce a pharmacological effect to a certain point?

Answer 5

The receptor becomes desensitised due to a genetic mutation, rendering the affinity of an agonist to be dysfunctional and unable to produce an effect

The receptors are already occupied

Tissues/ cells/ receptors have reached maximum capacity

Question 6

What does the concentration response curve show?

Answer 6

purpose: the response of the tissue at a given concentration of the agonist
Shape: sigmoidal curve (S shaped)
Can find the:
EC50 value (the concentration of the agonist at which 50% of the maximal response is produced) - which helps to determine the potency

Question 7

What is the EC50 value?

Answer 7

the concentration of the agonist at which 50% of the maximal response is produced) - which helps to determine the potency
Draw a horizontal at 50% of the maximal response to the curve, then from that point on the curve, draw a vertical line down to the x axis to find the concentration of the agonist
The concentration of the agonist tells you how potent the drug is
If the curve is shifted more to the left, it is more potent because this would mean less concentration of an agonist would be required to produce the same effect- 50% maximal response
If the curve is shifted more to the right, it is less potent, as more concentration of an agonist would be required to produce the same effect- 50% maximal response

Question 8

What is the logistic equation?

Answer 8

Helps to show the agonist concentration- response curve in equation form
Can be used to find: the hill slope (slope of the sigmoid) and the EC50 value (the concentration at which 50% of the maximum response will be obtained
Response = [agonist concentration] / ([agonist concentration] + equilibrium constant).
Note: doesn’t factor in the changes of the hill slope at different points, however, when response=50%, the agonist concentration is equal to the equilibrium constant which is the EC50

Question 9

Why is a simple two-state model better than the occupancy model of drug action?

Answer 9

The two-state model explains how some agonists generate a less maximal response than the other agonists (at the same receptor/ tissue)
→ This model helps to separate between how the agonist actually binds (affinity) and ability to stimulate a response (efficacy)- which is distinguished by the 2 separate pathways
Whereas, the occupancy model only shows the affinity and efficacy of the drug, but does not explain why some generate a high or lower maximal response
→ However, the occupancy model simply gives a generalised statement that affinity affects efficacy to produce a response (no specific pathways)

Question 10

Describe the process of the simple two-state model of agonist action?

Answer 10

Agonist (A) binds to the receptor (R )- [affinity state] to form the AR complex (intermediate phase) → this causes a change in the receptor structure (AR)
The change in the AR* receptor structure activities a series of second messenger, signalling cascades (chemical process that comprises at least two or more consecutive reactions)- which is the efficacy of the agonist
Finally, the a response occurs through the activation/ initiation of signal transduction pathways

Question 11

What are G protein Coupled receptors (GPCR)?

Answer 11

Alternative names: metabotropic/ 7 membrane-domain (7- TM ) receptors
Shape: 7 membrane-bound complex
Consists of: 3 subunits (α, β, γ)
There are 100s of different GPCR receptors but only a handful of G proteins

Question 12

What happens when a G protein-coupled receptor is activated?

Answer 12

1. Agonist binds to the G protein coupled receptor (GPCR, or R)
2. The G-proteins are released and freed (into an alpha subunit and beta-gamma βγ complex)
3. G proteins will then have an effect (i.e. either activate or inhibit enzymes or adjust the ion channel activity)
   → Usually the alpha subunit will activate an effector protein (denoted by E) (acts to execute the effects of signalling pathways, often as a response to external or internal signals) to produce second messenger molecules
   ** Some examples of effector proteins are= adenylate cyclase and phospholipase C**
   → Common G protein regulated signal transduction pathways: involve the (POSITIVE AND NEGATIVE) modulation of adenylate (AKA adenylyl) cyclase (to increase or decrease intracellular cAMP), or the ACTIVATION of phospholipase C (this makes two intracellular messengers, diacylglycerol and inositol trisphosphate, IP3).
   → Some G proteins that were directed and regulated by GPCR receptors can also activate other (monomeric- molecules that can react with other molecules to form very large complexes ) G proteins
4. The second messengers result in an effect on the cell (for example contraction, relaxation, secretion, neurotransmitter release, etc.)

Question 13

What are some examples of effector proteins?

Answer 13

adenylate cyclase and phospholipase C

Question 14

What are G-proteins?

Answer 14

Also known as guanine nucleotide-binding proteins

act as molecular switches inside cells, and are involved in transmitting signals from a variety of stimuli outside a cell to its interior.

specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Some G proteins, such as the signaling protein Ras, are small proteins with a single subunit.
Inactive: when it binds to GDP
Active: binds to GTP

Consists of: alpha subunit, Beta and gamma subunits (Alpha and gamma subunits have a tip, because they are lipophilic anchored to the membrane)

Some G proteins that were directed and regulated by GPCR receptors can also activate other (monomeric- molecules that can react with other molecules to form very large complexes ) G proteins

Question 15

How do G-proteins work?

Answer 15

In the resting/ basal state:
- 1. the G protein alpha subunit and the Beta-gamma (βγ) subunit are attached to each other with the guanosine diphosphate (GDP) bound to it
- 2. An agonist will bind to the receptor (pink) causing a conformational change in receptor structure which allows the G- alpha subunit to bind to the receptor
- 3. The G- alpha subunit will then undergo its own conformational change, and hence, releases GDP and allows GTP (guanosine-5’-triphosphate) to bind to the G- alpha subunit
- 4. By allowing GTP to bind to the G-alpha subunit, it splits the alpha- subunit complex and Beta-gamma subunit complex. This allows the 2 different sets of subunits to elicit different effects on targets
- 5. The G-alpha subunit then binds to a effector molecule (Target 1), which makes the target activated and converts GTP to GDP (+ phosphate- P, which inactivates the alpha subunit.
→ target/ effector molecule relay signals via 2nd messenger
-6. Since the G-alpha subunit (bound to GDP) is inactive, it will try to bind to the Beta- gamma subunits, and by doing so, inhibits the activity of the Beta-gamma subunit from doing what they were intended/ were doing

Note: Despite the beta-gamma subunits being prevented from performing its activity, as long as there is an agonist present, this cycle/ steps will occur

Question 16

What is the difference between GDP and GTP?

Answer 16

GDP is the product of GTP dephosphorylation by GTPases, e.g., the G-proteins that are involved in signal transduction.

GDP is converted into GTP with the help of pyruvate kinase and phosphoenolpyruvate.
This is why GDP is stored and then released so that it can be used

Question 17

Example of a peptide hormone signal transduction via a G-protein coupled receptor:

Answer 17

Peptide hormones are usually short chain of amino acids, usually water-soluble and too large to pass the lipophilic membrane of the target cell
Interacts with a hormone receptor that has an exposed extracellular hormone binding site
Peptide hormone fits into the receptor
Activated receptor goes under a conformational change→ allowing the hormone messages to enter the cell
Once the hormone messages are in the cell, they are converted/ transduced into chemicals, known as second messengers, which control the cell’s response (i.e. depolarization or hyperpolarization of a cell)

Question 18

What do GPCRs control/ modulate in the cell?

Answer 18

Enzyme:
→ Adenylyl cyclase: membrane bound enzyme responsible for cyclic adenosine monophosphate (cyclic AMP or cAMP) formation
→ phospholipase C: responsible for inositol phosphate and diacylglycerol (DAG) formation
Ion channels:
→ calcium and potassium channels
Systems:
→ Rho A/ Rho kinase: regulates the activity of many signalling pathways, such as those controlling cell growth and movement
→ Mitogen-activated protein kinase (MAP kinase): controls the many cell functions including cell division

Question 19

What is the Adenylyl cyclase/ cAMP system?

Answer 19

Responsible for the production of cAMP (a 2nd messenger pathway) from the substrate ATP
When adenylyl cyclase is activated by the GTP-bound alpha subunit (active form of the G-protein), catalyses synthesis of the second messenger cAMP from ATP. Since adenylyl cyclase are activated by GPCR (G alpha subunits), they can also be membrane bound
Due to the positive/ negative regulation of the adenylyl cyclase (switching on and off), it helps regulate the production of cAMP

Question 20

How is adenylyl cyclase bidirectional?

Answer 20

Due to the G proteins:
→ G-alpha S proteins (Gαs)- stimulates, switches adenylyl cyclase on
→ G-alpha i proteins (Gαi)- inhibit, switches adenylyl cyclase off

Question 21

What is the role of cAMP?

Answer 21

Activates protein kinase A (PKA)
Role: phosphorylate cellular proteins/ enzymes, activating or inhibiting the proteins
PKA can have multiple effects inside the cells, from the inhibition of smooth muscle contraction to increasing cardiac muscle activity as well as changes in cell transcriptomic activity.
PKA will phosphorylate certain serine or threonine amino acids. As it happens, the intracellular parts of voltage gated calcium channels (present in, for example, neurons and cardiomyocytes) contain a number of serine and threonine residues. Phosphorylation of these by PKA potentiates the activity of those channels.
When cAMP is increased, activates the PKA, PKA phosphorylates the enzymes and the transcription factor: CREB (cAMP, responsive, element binding protein) gets phosphorylated and then diffuse into the nucleus and binds to the CRE, which is a cAMP promoter of the DNA and starts the transcription of the particular DNA sequence

Question 22

What is the phospholipase C/inositol phosphate system?

Answer 22

Phospholipase C (PLC) produces inositol trisphosphate (IP3) and diacylglycerol (DAG) from the splitting (cleavage) of phosphatidylinositol bisphosphate (PIP2)
Note: PIP2 can be cleaved by a range of different molecules, which will ultimately produce active mediators (i.e. IP3 and DAG)
E.g. PIP2 cleavage by Ca2+ dependent phospholipase A2 leads to formation of arachidonic acid and subsequent prostaglandins / thromboxane production.
PIP2 cleavage by phospholipase D creates another messenger, phosphatidic acid.
Figure above: Structure of phosphatidylinositol bisphosphate (PIP2), showing sites of cleavage by different phospholipases to produce active mediators

Question 23

Why is PIP2 important in cellular signalling?

Answer 23

It is the important molecule that produces second messengers in order for a pharmacological effect to occur, including IP3 and DAG and many more

Question 24

How does phospholipase C/inositol phosphate system produce IP3 and DAG?

Answer 24

GPCRs will bind to Gαq proteins
Gαq proteins will activate the PLCβ (Beta version of the Phospholipase C- PLC), which causes the splitting/ cleavage of PIP2, forming DAG and IP3

Question 25

What is the role of inositol trisphosphate (IP3) and how ?

Answer 25

Activates ion channels on calcium storage areas to increase the intracellular calcium level
Process:
IP3 activates (ligand gated) IP3 receptors on the sarcoplasmic reticulum- in skeletal muscles (SR), which releases Ca2+ ions

Question 26

What is the role of the membrane bound diacylglycerol (DAG) and how?

Answer 26

Activates the protein kinase C (PKC)
Process:
DAG (often combined with elevated levels of intracellular calcium) recruits and activates certain isoforms of PKC (protein kinase C)

Question 27

How does the release of Ca2+ ions help with function?

Answer 27

Calcium can activate things on its own OR:
Activate things via intermediate proteins such as calmodulin

Question 28

What are the different ways in which protein kinase C (PKC) can be activated?

Answer 28

Activated by GPCRs:
- Conventional (activated by calcium and DAG) : Below is the general domain structure of conventional PKC, includes- Regulatory domain and amino terminus (contains N end that is connected to amino molecules) and Kinase domain and carboxy terminus (contains the connected conventional PKCs- alpha, beta and gamma)

B) Novel (activated by DAG, not calcium))
C)
Not activated by GPCRs:
Atypical (not activated by DAG or calcium)

Question 29

What happens to protein kinase C at rest?

Answer 29

The N-terminus of PKC (pseudosubstrate domain) is “stuck” in its catalytic domain (the domain in which it can be catalysed)
The Ca 2+ ions that are released gets bound to the C2 domain (yellow), which activates the conventional PKC
→ Since Ca2+ ions bind to the C2 domains, this creates contact with anionic phospholipids (especially phosphatidylserine) in the cell membrane
Once Ca2+ bound, the PKC C1a domain is now available for binding to DAG.
→ Binding of the C1 domain(s) to their appropriate lipid partners releases the pseudosubstrate from the kinase-binding pocket leading to kinase activation.

Conventional PKCs are in the cytosol, that wait for the increase of intracellular Ca 2+ ions to trigger them
Resting cellular Ca2+ ~100 nM is not enough to get conventional PKCs to distribute to the membrane from the cytosol

Question 30

What is membrane association?

Answer 30

Refers to the interaction and attachment of proteins, lipids, or other molecules with the cell membrane

Question 31

What is membrane association?

Answer 31

Refers to the interaction and attachment of proteins, lipids, or other molecules with the cell membrane

Question 32

What is the membrane association in PKCs?

Answer 32

Refers to the interactions and attachments of proteins, and other molecules within the PKCs to drive a function

Question 33

What is the membrane association in PKCs driven by?

Answer 33

Ca2+ ions binding to the C2 domain, which contacts anionic phospholipids

Question 34

What does DAG-C1 binding cause?

Answer 34

DAG-C1 binding might occur after Ca2+ binds to the C2 domain, which causes the C1 alpha domain to be available for DAG to bind to C1 domain
This DAF-C1 binding results in;
→ pseudosubstrate (green) dissociation
→ kinase activation
→ holding the active enzyme up at the plasma membrane increases substrate contact

Question 35

How many receptors do cells contain?

Answer 35

Cells contain multiple G protein-coupled receptors and often have receptors that help activate and then deactivate (pro- and anti- contraction receptors) to maintain balance
So, the cells often receive mixed messages about what to do
→ E.g. So all smooth muscle cells contain pro- and anti-contraction receptors!!! in smooth muscles:
activating Gαs coupled GCPRs means smooth muscle relaxation= opposing muscle contraction while activating Gαq/11/14/15 coupled GCPRs means smooth muscle contraction.

Question 36

What is a common role of PKA and PKC?

Answer 36

PKA and PKC can phosphorylate GPCRs and ion channels to modify their activity