Basic Principles of cell signalling and GPCR system

Question 1

What is Signal Transduction?

Answer 1

= conversion of one signal into another

• involves growth factors, cytokines, hormones, ECM, neurotransmitters, light, sound, etc.
• controls all aspects of normal development and physiology. so if it doesn’t work, it’s the initiator to disease
• Key players: RTKs, GPCRs, Ras, MAPK

Question 2

Basic elements of cell signaling

Answer 2

• Extracellular Signal or Signalling molecule (ligand or primary messenger)
- small molecules (epinephrine, acetylcholine, steroids, peptide hormones, monoamines AA, etc.)
- large molecules(eg, hydrophilic), growth factors, cytokines (proteins)
• Receptor Protein
- Cell-surface (most common)
- Intracellular
• Intracellular signalling and effector proteins
- G proteins, protein kinases, and phosphates, etc
- they elicit a phenotype, have an effect on cell (ex, metabolic enzyme for altered metabolism, gene regulatory protein for altered gene expression, cytoskeletal protein for altered cell shape or movement)
• Second Messengers (Ca2+, cAMP, cGMP, IP3, DAG, NO, etc.)

Question 3

Overview of cell signalling

Answer 3

MEdiated through sequential protein-protein interactions.
- you have hydrophobic molecule (eg, steroids) that cross cell pm and bind to cytosolic intracellular receptor. This complex goes and modifies gene expression and development
OR
- you have a hydrophilic molecule (eg, small molecules, peptides, proteins) that can’t cross cell membrane, so it uses growth factor to bind to cell surface receptor —> the binding causes a conformational change in receptor which causes wiggling —> signal transduction proteins and second messengers are recruited and act —> Effector Protein activated and inhibits cell-surface receptor + indirectly does Modification of cellular metabolism, function, movement + Modification of gene expression and development

Question 4

Hydrophobic Signalling using Nuclear-receptor superfamily (specific example: Glucocorticoid Receptor)

Answer 4

• All these Nuclear-receptors (Intracellular receptors) have a Variable region (activation Domain AD), a DNA-binding domain (can alter gene expression), and a Ligand-binding domain LBD (binds the hydrophobic molecule)
• Glucocorticoid Receptor (GR) is found in cytoplasm and is inactive. The hormone crosses cell membrane and binds LBD of GR which removes Inhibitor HSP90, which now allows GR to move into Nucleus so that it can interact with the Response Element there (which is where GR’s AD further increases the expression of genes which have the GR response element further upstream)

Question 5

4 Forms of Intracellular signalling

Answer 5

Endocrine signalling: ex, Insulin (extracellular signal) can be produced in one type of cell, act in the endocrine system by being secreted into blood by endocrine gland, and then act on distant target cells

Paracrine signalling: Secretory cell ejects extracellular signal which goes into adjacent target cell

Autocrine signal: Secretory cell ejects extracellular signal to receptors (target sites) on same cell

Signalling by plasma membrane-attached proteins: Signalling cell (has membrane-attached signal) binds to adjacent target cell that has the receptor

Question 6

True or False: Signalling molecules can act locally or at a distance

Answer 6

True.

• some signalling molecules can act in both endocrine and paracrine signalling (eg, epinephrine)
• some can act by cell-cell, autocrine, or paracrine signaling (eg, epidermal growth factor EGF)

Question 7

Signalling by cell-surface receptors

Answer 7

1. Synthesis and Release (close or far) of the signaling molecule / ligand by the signalling cell
2. Transport and binding the signal to a specific receptor of the target cell
3. Conformation change of receptor
4. Initiation of one or more intracellular signal-transduction pathways
   5a. Short-term cellular responses (ie, modification of cellular metabolism, function, movement)
   5b. Long-term cellular responses (ie, modification of gene expression, development) – more common
5. Termination of cellular response – if you don’t ==>cancer

Question 8

Extracellular signals can act slowly or rapidly to change behaviour of target cell

Answer 8

Fast:

• < sec to mins
• altered protein function leads to —> changes in ion transport, cell movement, secretion or metabolism
• ideal in brain… don’t want intermediates

Slow:

• mins to hrs
• goes into nucleus to alter protein synthesis/translation which leads to —> gene regulated changes in cell growth and division
• both then lead to altered cytoplasmic machinery —> then altered cell behaviour

Question 9

What kinds of extracellular signals is a cell exposed to?

Answer 9

• multiple for just “Survive”
• mitogenic signals on top of survivals signals for “grow and divide”
• “differentiate” signals on top of survivals signals
• removal of all signals for apoptosis

Question 10

LigandReceptor Interactions

Answer 10

• binding specificity is based on the Molecular Complementarity of the surfaces of the receptor (binding interface) and the ligand (noncovalent forces)
• this binding triggers a conformational change in the receptor
• very often the ligands (signalling molecules) will induce Receptor Dimerization (aka, will recruit another receptor)

Question 11

The Dissociation Constant

Answer 11

R + L <=> RL(receptor-ligand complex)
- to measure how compatible they are use Kd
- Kd = [R][L]/[RL]
= the ligand concentration required to bind 50% of the cell surface receptors
= the measure of affinity of a receptor to its ligand
- if you have a high affinity ==> not a lot of ligand required to occupy the 50%
- you don’t want a promiscuous ligand, you want one that needs a little only

Question 12

How do you use Functional expression assay to identify a cDNA encoding a cell surface receptor?

Answer 12

• the cultured cells do not express correct receptor for ligand X == non-responding cells, therefore no binding of ligand to intracellular receptor, no cellular response
• isolate the RNA from a responding cell, convert it to cDNA, integrate it into plasmids to create a library
• transfect the non-responding cells with the cDNA expression vector + growth factor (Now this cell expresses the proper receptor), and screen for cellular phenotype associated with ligand X (for example, proliferation)
• identify incorporated cDNA by PCR followed by sequencing. The receptor protein can be deduced from the cDNA sequence.
• And you can put that cDNA into another cell to see if indeed that protein is for this
• also can mutate different AAs to see what the essential binding domain is

Question 13

Regulation of protein activity by a kinase/phosphatase switch

Answer 13

• posttranslational modification
• kinases put P groups on proteins, phosphatases take off
• 3 types of AAs that are de/phosphorylated(Tyrosine, Serine, Threonine)
• phosphorylated substrate proteins are active, a protein phosphatase will de-P the protein, thus inactivating it
• then protein kinase using ATP, adds a P group

Question 14

Switching Mechanism of G protein

Answer 14

• posttranslational modification
• G proteins can be active (bound to GTP) or inactive (bound to GDP)
• GTP-bound actually has a different conformation and stabilized and “switch I and II” are there
• activation is triggered by a signal (ie, hormone binding to receptor) and is helped by GEF (activator protein) binding to G protein to remove GDP. Then GTP is bound which leads to activation.
• to inactivate, GTPase mediates it either by intrinsic activity (the proteins cut off GTP themselves) or with separate proteins (using GAP)
• GTPase Activator Protein (GAP) can accelerate GTP hydrolysis to GDP (going to inactive state)

Question 15

G protein-coupled receptor system Overview

Answer 15

= a receptor with 7 membrane-spanning domains
+ coupled trimeric G proteins
+ a membrane-bound effector protein
- they don’t bind GTP or GDP directly, they just couple, and control a number of processes
- they’re a second messenger in many GPCR pathways

Question 16

G protein-coupled receptors in G protein-coupled system

Answer 16

800 different types of GCPRs but all have:

  - 7 transmembrane domains (H1-H7) 
  - 4 extracellular domains (E1-E4) -- to bind ligands (light, hormones, NTs, chemoattractants, odorants, etc.)
  - 4 cytosolic domains (C1-C4) -- involved in how different intracellular proteins associate

Question 17

What is the major target of pharmaceutical drugs?

Answer 17

Agonists and antagonists

I think in relation to G protein-coupled receptors

Question 18

Trimeric G protein in G protein-coupled system
Overview of structure
Function
Mechanism of Action

Answer 18

• in the GTPase superfamily of G proteins, “G” refers to the ability to bind guanine nucleotides (GDP or GTP)
• 3 subunits (Galpha, Gbeta, Ggamma) but we usually call it Gbetagamma because they never dissociate
• Galpha and Ggamma are proteins that are lipid-anchored to the pm (!!) at the cytoplasmic face
• all the different variations of Gbgs usually have same function, while Gas have variety
• Galpha-GTP is active
• Function: in signal transduction by coupling ligand-bound receptors to specific effector molecules

Question 19

G protein-coupled system

Answer 19

1. Binding of hormone(ligand) to G protein receptor induces a conformational change in which the cytoplasmic domains have changed to be able to bind Galpha
2. Activated receptor binds to Galpha subunit of G protein
3. Activated receptor causes conformational change in Galpha, triggering dissociation of GDP from it
4. Binding of GTP to Galpha triggers dissociation of Galpha from the receptor and from Gbetagamma
5. Hormone dissociates from receptor. Galpha binds to effector, activating it (short-lived state– on for short then off). (Sometimes effectors can act as GAP – to help hydrolyze GTP to GDP)
   *** note: sometimes Galpha inhibits effector and Gbetagamma activates it
6. Hydrolysis of GTP–> GDP causes Galpha to dissociate from effector and reassociate with Gbetagamma
   Now you’re back at the state of inactive receptor, inactive effector, with full trimeric G protein

Question 20

How can you demonstrate receptor-mediated dissociation of Galpha or Gbetagamma?

Answer 20

FRET = Fluorescence Resonance Energy Transfer

• Link CFP to alpha subunit, and YFP to gamma. They interact as long as alpha subunit is bound to GDP!
• Shine excitation light of 400nm on CFP. CFP emits its light which (energy transfer) lights up YFP to emit fluorescence of 527nm in yellow
• YFP won’t light up when Ga and Gbg are dissociated (aka, when GTP is bound), only CFP will (490nm)