Transduction mechanisms

Question 1

Name as many ligand categories as you can.

Answer 1

• inorganic / organic compounds
• amino acids/ biogenic amines
• small peptides
• proteins
• lipids
• steroids and fatty acid derivatives
• nucleotides
• small gases (e.g. nitric oxide and carbon monoxide)
• photons (light)

Question 2

How much of human genome encodes for proteins involved in signal transduction ?

Answer 2

> 20%

Question 3

How much of human genome encodes for GPCRs ?

What about in C. elegans ?

Answer 3

~ 1% (1000-2000)

In C. elegans this is > 5% of the genome).

Question 4

How does the speed at which an external signal is converted in to a biological response vary ?

Answer 4

The speed at which an external signal is converted in to a biological response varies depending on the type of transduction cascade utilised by its receptor.

Question 5

What are LGICs ?

Give an example of LGIC you know.

Answer 5

LGICs = ultimeric proteins which combine the functions of receptor and effector into a single macromolecule
e.g Nicotinic acetylcholine (nAChR) receptor: five subunits arranged symmetrically around a central pore 
(290kDa). 
17 nAChR subunits have been identified :
~ 10 isoforms of  α subunit
~ 4 isoforms of β subunit
gamma subunit
delta subunit
epsilon subunit

Question 6

What is the advantage of having many different channel subunits ?

Answer 6

Many different subunits = many different flavours of the channel –> plasticity

Question 7

What is the effect of nAChR activation in muscle ?

Answer 7

For the muscle nAChR the inward current causes depolarization of the muscle membrane and initiates a muscle AP within one millisecond of the binding of ACh to the receptor.
No other proteins are involved in generating the response.

Question 8

What kind of transduction mechanisms are LGICs useful for ?

Answer 8

Ligand gated ions channels mediate rapid information transfer.

Question 9

What are the general characteristics of GPCRs ?

Answer 9

• The most common type of plasma membrane receptor
• Named because they must interact with an intracellular protein complex, known as a GTP-binding protein or G-protein, in order to produce a response.
• Amongst the oldest of the signal transduction mechanisms
• ~1% of human genome enocdes for these receptors
• Are the target for >50% of current therapeutic drugs

Question 10

Do all GPCRs show sequence homology ? - anything in common ?

Answer 10

Sequence comparison have revealed different receptor families with no obvious sequence homology, but all share a common monomeric heptahelical structure.
All have the same basic structure comprising a single peptide chain containing 7 transmembrane alpha-helices

Question 11

How many families of GPCRs are known ?

Answer 11

Five main families of GPCRs have been re-classified on the basis of phylogenetic criteria.

Question 12

What are G-proteins ?
What are the different subunits ?
What are their respective roles ?

Answer 12

G-proteins are transducing elements composed of three subunits termed α, β and gamma.
The α subunit binds guanine nucleotides and has enzymatic activity catalysing the conversion of GTP to GDP.
The beta and gamma subunits associate tightly into a complex and can anchor them selves into the plasma membrane through a lipid modification (prenylation) of the gamma subunit.

Question 13

What are the 3 proteins involved in GPCR signal transduction ?

Answer 13

• Receptor = GPCR
• G-protein
• Effector = e.g. AC, PLC, etc.

Question 14

What are the different steps of GPCR signal transduction ?

Answer 14

1. Ligand binding
2. Conformation change in receptor
3. Interaction of the alpha subunit of the G-protein w/ the receptor (via the 3rd intracellular loop)
4. Binding of the alpha subunit to the activated receptor triggers exchange of GDP for GTP
5. The activated G-protein dissociates from the receptor and seperates into alpha and beta/gamma subunits
6. The GTP bound alpha subunit interacts with the effector protein (AC)
7. Activation of AC and conversion of ATP to cAMP
8. Hydrolysis of GTP to GDP by the alpha subunit, which leads to dissociated from its effector.
9. The alpha and beta/gamma subunits re-associate –> the response is terminated

Question 15

How may hydrolysis of GTP to GDP be accelerated ?

Answer 15

Hydrolysis of GTP to GDP may be accelerated by other

proteins that act as GTPase-activating proteins (GAPs), specific for Gα subunits, e.g. Regulators of G-proteins (RGS).

Question 16

Can the effector itself possess intrinsic GAP activity ?

Answer 16

Yes, e.g. PLCβ

Question 17

How do the time-scales for smooth muscle contraction elicited by different routes for Ca2+ entry differ ? (L/VGIC Vs GPCR)

Answer 17

L/VGIC = 0.3 s
GPCR = 1.3 s

Question 18

What are the different G-proteins you know ?
Alpha subunits are they coupled to ?
What are their functions ?
By which bacterial toxins are they modified ?

Answer 18

Gs –> αs = activates AC + Ca2+ channels, activated by cholera
Gi –> αi = inhibits AC + activates K+ channels, inhibited by pertussis
Gq/11 –> αq/11 = stimulates PLCβ + activates K+ channels
G12/13 –> α12/13 = regulates Rho family GTPase signalling + cytoskeleton remodeling
Gt –> αt = activates cGMP PDEs in vertebrate rod photoreceptors, activated by cholera + inhibited by pertussis
Go –> αo = stimulates PLCβ, activates K+ channels + inactivates Ca2+ channels, inhibted by pertussis

Question 19

How many different G-protein subunits are known ?

Answer 19

> 20 α subunits
~4 β subunits
~7 gamma subunits

Question 20

Give examples of receptors (and their ligands) that couple to Gs, Gi/Go, abd Gq.

Answer 20

Gs : 
- NA --> β-AR 
Gi/Go : 
- Ach --> M2-musc
- NA --> α2-AR
- Enkephalin, morphine --> mu/delta - opiate
Gq : 
- NA --> α1-AR
- ACh --> M1-musc

Question 21

Give examples were NA and ACh have either antagonistic of synergistic effects.

Answer 21

Heart –> NA (Beta, Gs) + ACh (M2, Gi) = antagonistic

Smooth muscle –> NA (alpha 1, Gq/11) + (M1, Gq/11) = synergistic

Question 22

What are the consequences of the GPCR transduction pathway ?

Answer 22

• Operate on a slower time scale compared to LGICs
• Have built-in amplification
• Variety of G-protein isoforms allow coupling of different receptors to many different effectors

Question 23

Name four classes of enzyme linked receptors.

Answer 23

Receptor tyrosine Kinases (RTKs)
Receptor guanylyl cyclases (e.g. ANP receptors)
Receptor serine/threonine kinases
Tyrosine Kinase associated receptor (have no intrinic kinase activity but are closely associated with an intracellular kinase)

Question 24

How many RTKs are known ? - how many families ?

In what form are most found ?

Answer 24

> 90 RTKs distributed into ~ 20 families

Most are single subunit receptors but some exist as complexes e.g., the insulin receptor

Question 25

Explain how RTKs work, with the example of the platelet derived growth factor receptor (PDGFR).

Answer 25

1. PDGF binding leads to receptor dimerization
2. A conformational change renders the active sites w/in the kinase domain available to bind ATP, leading to autophosphorylation of tyrosine residues
3. The phosphorylated tyrosine residues act as docking sites for effector molecules containing src homology domains (SH2, SH3 domains).
4. SH2 domains specifically recognize the phosphorylated state of tyrosine residues, thereby allowing SH2 domain-containing proteins to localize to tyrosine-phosphorylated sites (> 115 proteins with SH2 domain have been identified within the human genome)
5. Different effector can interact w/ specific tyrosine residues of the activated receptor (e.g. PLCgamma)

Question 26

What are 3 examples of RTKs you know ?

Answer 26

• PDGFRa
• FGFRs
• Eph Rs

Question 27

What are the main characteristics of enzyme linked receptors ?

Answer 27

• Most are single membrane spanning proteins
• They dimerise upon ligand binding leading to auto-phoshphorylation of tyrosine redisues
• Phosphorylated tyrosine residues act as recognition sites for proteins with SH2/3 domains
• Many different effectors may interact with the same receptor
• Responses operate on the minutes/hours time scale

Question 28

What are the main properties of intracellular DNA binding receptors ?

Answer 28

• These receptors are generally in the cytoplasm or nucleus
• Their ligands are membrane permeable (e.g. steroid hormones –> time course for steroid action is typically slow)
• The receptors have ligand binding, DNA binding and transcription activating domains