How many GPCR genes are known to exist?
>900 in man.
What is the largest family of related proteins?
What component of GPCRs has been conserved throughout eukaryotes?
Give examples of the different stimuli GPCRs bind.
Give examples of the functions of GPCRs.
- Cell growth
What percentage of prescribed drugs act at GPCRs?
>30% - hundreds more in late stage development
Give examples of prescribed drugs that act at GPCRs
- Salbutamol (ß2-adrenoceptor agonist - asthma)
- Morphine (µ/k opiate receptor agonist - analgesic)
- Losartan (AT1 receptor antagonist - hypertension)
In the structure of GPCR, what does the common core domain consist of?
Seven membrane-spanning a-helices
Where in the GPCR structure are the N-and-C-terminus located?
- Extracellular N-terminus
- Intracellular C-terminus
How many intracellular and extracellular loops are the a-helices connected by in the GPCR structure?
3 Intracellular & 3 Extracellular loops
What are intracellular loops involved in?
G-Protein coupling & regulation of signalling
How many components do GPCR signalling systems have and what are they called?
What do the G-Protein and Effector do in the singalling systems?
Translate agonist binding event into generation of second messenger.
How many families can GPCRs be divided into and what are their names?
- Family 1a, 1b, 1c
- Family 2
- Family 3
- Family 4
- Family 5
[Families 1,2&3 are the largest group]
How are the families of GPCRs distinguishable?
Each family is distinguished by the mechanism of receptor activation by the agonist.
Describe the agonists invoved in GPCR Family 1a & give examples.
Small molecule agonists.
Ligand binds in cavity in common core region.
e.g Epinephrine, Nucleosides.
Describe the agonists involved in GPCR Family 1b & give examples.
Short peptide agonists.
Peptide ligand binds to N-terminal domain & residues in extracellular loops.
C-terminus of peptides also bind cavity in common core.
e.g Thrombin, Chemokines
Describe the agonists involved in GPCR Family 1c and give examples.
Ligand binds to very large N-terminal domain.
N-terminal domain then contacts extracellular loops to activate.
e.g Thyroid Stimulating Hormone (TSH)
A detailed crystal structure of which family of GPCRs is now available?
>10 Family 1a GPCRs
e.g ß1 and ß2 adrenoceptors
In the GPCR structure, what do helices form?
A cavity/pocket responsible for ligand binding
Describe the cavity in the GPCR structure.
Largely non-polar with a few critical polar residues responsible for specific high-affinity interactions with ligand.
What core structure is common to all GPCRs?
What 3 structural features are common to Family 1 GPCRs?
- Cysteine residues in extracellular loops 1 & 2
- Proline residues in TM helices 6 & 7
- Asp in TM2 and AspArgTyr ("DRY") in second intracellular loop
In Family 1 GPCRs, what is the function of the cysteine residues in extracellular loops 1 and 2?
They form a disulphide link important for receptor stability & helical packing.
In Family 1 GPCRs, what is the function of the Proline residues in TM helices 6 and 7?
They introduce kinks into the a-helices, and pivots for conformational change.
In Family 1 GPCRs, what is the function of the Asp in TM2 and "DRY" in second intracellular loop?
Important for receptor activation of G-Protein.
What controls the activity of the effectors after activation by GPCRs?
G-Proteins (Guanine Nucleotide-Binding Regulatory Proteins)
How many different subunits do G-Proteins have and what are they?
3 - They are heterotrimeric.
a, ß and Y.
How are G-Proteins attached to the membrane?
By lipid modifications on a & Y.
Which subunit can bind GDP or GTP and possesses GTPase activity, all of which is critical for function?
What states to G-Proteins cycle between?
GDP-Bound Inactive state & GTP-Bound Active state.
What is the state called when there is no activity in the GPCR structure i.e it is at rest?
When the agonist binds to the receptor, what does it attract?
The agonist-bound receptor attracts GDP-bound G-Protein.
What does the receptor-bound G-protein release?
When GDP is released, what then can happen?
GTP can now bind to G-Protein.
When GTP binds to the G-Protein, what happens?
The G-protein dissociates into GTP-a and ßY complex.
When the GTP-a and ßY complex forms, what happens?
GTP-a and ßY bind and activate different effectors.
When the effectors are bound by GTP-a and ßY, what happens?
GTP is hydrolysed to GDP on the a-subunit
When GTP is hydrolysed to GDP on the a-subunit, what happens?
GDP-a and ßY dissociate from the effectors, inactivating the effectors.
When the effectors are inactivated, what happens to the 3 subunits?
The G-Protein trimer reforms, and the basal state resumes.
What does the agonist-induced movement of helices do?
What sequence of events causes the G-Protein to be activated by agonist-induced movement of helices?
- TM's 3 & 6 move away from each other
- TM6 rotates on its own axis by 30°
- Opens cleft for C-terminus of G-protein a-subunit
- Ga-subunit activated by interaction with DRY & other sequences
Can distinct effectors only be regulated by one G-Protein?
No, multiple G-proteins can regulate distinct effectors.
How is each G-protein classified?
By the identity of the a-subunit
What effectors does Gs regulate and how?
- Increases Adenylyl Cyclase
- Increases Ca2+channel
What effectors does Gi regulate and how?
- Decreases Adenylyl cyclase
- Increases Potassium channel
What effectors does Gq regulate and how?
- Increases Phospholipase C
What effector does Gt (transducin) regulate and how?
- Increases cGMP phosphodiesterase
Give an example of a GPCR which activates multiple G-Proteins.
Thrombin activation of PAR-1 in vascular endothelium.
[PAR-1 = Protease-Activated Receptor-1]
Which G-Proteins are activated by thrombin activation of PAR-1 and what effects does this cause?
These cause :
- Secretion of NO/PGI2/EDHF/TXA2
Why is activation of multiple signalling pathways necessary?
To initiate complex biological phenomena such as angiogenesis (new blood vessel formation from pre-existing vessels)
What facilitates signal amplification?
The design of the GPCR signalling cascade.
Describe the signalling cascade using the example of Rhodopsin signalling in rod cells in the retina.
1. 1 Rhodopsin absorbs 1 photon
2. 500 Transducin molecules activated (G-PROTEIN)
3. 500 Phosphodiesterase molecules activated (EFFECTOR)
4. 10,000 cGMP molecules hydrolysed
5. 250 channels close
6. 107 Na+ ions/sec prevented from entering cell
→ Hyperpolarisation of 1mV at rod cell membrane.
How do GPCR responses differ from that of LGIC?
Use ACh binding to illustrate.
Slower & more sustained.
ACh binding to Nicotinic receptor → Immediate opening of ion channel.
ACh binding to Muscarinic receptor → Activation of 2 more proteins needed to give signal(Gi-Protein and K+ channel effector) - Slower onset.
In GPCR, what causes the release of a second messenger, and what then happens?
- The effector protein, once activated by the G-Protein, releases a second messenger which then activates a second messenger-dependent kinase.
- This allows substrates to be phosphorylated, and so a change in activity occurs.
In the ß-adrenoceptor activation of cAMP signalling pathway, name the specific hormone, G-Protein, Effector protein, 2nd messenger and protein kinase involved.
- Hormone = Epinephrine
- G-Protein = Gs
- Effector = Adenylyl Cyclase
- 2nd Messenger = Cyclic AMP (cAMP)
- Kinase = cAMP-dependent protein kinase
What is the active cAMP-dependent protein kinase also known as, and what is its' function?
Also called PKA.
Responsible for most of cAMP's effects.
Adds phosphate group to serine or threonine residues on target proteins.
What does the structure of inactive PKA consist of?
- 2 Regulatory subunits (bind cAMP)
- 2 Inactive catalytic subunits (kinase activity)
When 4 cAMP molecules are added to PKA, what does the structure of active PKA consist of?
- Complex of cyclic AMP & regulatory subunits
- Released active catalytic subunits
When cAMP is added to PKA, what ensures high sensitivity to cAMP?
Positive cooperative binding.
Where is PKA Type I found?
In the cytosol
Where is PKA Type II found?
Tethered to specific subcellular locations via R subunit interaction with specific localisation proteins (A Kinase Anchoring Proteins: AKAPs)
Give an example of important PKA substrates in the liver in response to increased cAMP by glucagon.
- Phosphorylation & activation of phosphorylase kinase
- Phosphorylation & inactivation of glycogen synthase
Leads to breakdown of liver glycogen into glucose.
Glucose released into blood.
Give an example of an important PKA substrate in adipose tissue in response to increased cAMP by glucagon & epinephrine.
- Phosphorylation & activation of triglyceride lipase
Leads to breakdown of triglycerides which causes increased fatty acids & glycerol.
Give an example of an important PKA substrate in smooth muscle in response to increased cAMP by epinephrine.
PKA phosphorylates multiple substrates to relax smooth muscle.
What controls contraction in the myosin light chain (MLC)?
What is a more complex pathway than PKA pathway, and why?
Inositol 1,4,5 triphosphate/diacylglycerol pathway.
Multiple 2nd messengers generated from a single precursor at the plasma membrane.
What is the generic structure of a glycerophospholipid?
Glycerol backbone attached to 2 fatty acids and a phosphate group.
The phosphate group has an alcohol group attached to it.
How many second messengers does hydrolysis of PIP2 (phosphatidylinositol-4,5-bisphosphate) generate?
Two distinct second messengers.
What does the structure of PIP2 consist of?
- 1,2-Diacylglycerol (DAG) - Glycerol & 2 fatty acids
- Inositol-1,4,5-triphosphate (IP3) - Phosphate linked to glycerol & inositol-4,5-bisphosphate
What hydrolyses PIP2?
Phospholipase C, into DAG & IP3
What happens to DAG & IP3 when hydrolysed?
- DAG (Hydrophobic) - Remains at plasma membrane
- IP3 (Hydrophilic) - Diffuses into cytoplasm
What hormone-induced release does IP3 mediate?
Hormone-induced release of Ca2+ from the ER
What does emptying of the ER stores trigger?
Opening of store-operated Ca2+ channels at plasma membrane
What does Ca2+ bind to and where?
Protein kinase C (PKC) in the cytosol
Where does the Ca2+-bound PKC travel to?
Translocates to the plasma membrane to bind DAG & become active.
What makes calcium an ideal intracellular messenger?
1. Rapidly mobilised and removed:
- Steep gradient across plasma membrane, which allows for rapid increase of calcium into cell.
2. Binds tightly to target proteins:
e.g Calmodulin: Ca2+ can coordinate 6-8 oxygen atoms from:
- Glu, Asp side chains
- Gln, Asn side chains
- Main chain carbonyl C=O
By linking multiple regions of a protein, Ca2+ binding induces significant conformational changes.
3. Ca2+ mobilisation linked to receptor activation
What is the major [Ca2+]in sensor in eukaryotic cells?
When is CaM activated?
Binds Ca2+ & is activated when [Ca2+]in exceeds 0.5µM.
[Ubiquitously expressed & highly conserved from yeast to man].
How many binding sites does CaM have?
17kDa protein with 4 Ca2+ binding sites.
What type of binding is Ca2+ with calmodulin?
Ca2+ binding is positively cooperative
i.e small increase in Ca2+ leads to a large change in CaM activity.
What are 4 downstream binding partners for Ca2+/CaM?
- CaM Kinases
Give an example of a pump that is a downstream binding partner for calcium/CaM and descibe its mode of action.
- Activation helps reduce [Ca2+]in back to basal levels - Negative Feedback.
Give 2 examples of an enzyme that is a downstream binding partner for Ca2+/CaM and describe its mode of action.
Nitric Oxide Synthases (NOS):
- Forms nitric oxide (NO) from L-Arg
- NO = Important inter/intracellular messenger
Myosin Light Chain Kinase (MLCK):
- Mediates smooth muscle contraction in response to increased Ca2+
Give an example of a CaM Kinase that is a downstream binding partner for Ca2+/CaM and describe its mode of action.
- CaM Kinase = Family of multifunctional protein kinases
- Divided into 4 classes (1a, 1b, II and IV)
- Ubiquitous, but enriched at synapses
Give an example of a calcineurin that is a downstream binding partner for Ca2+/CaM and describe its mode of action.
- Ca2+/CaM-activated protein Ser/Thr phosphatase
- Controls phosphorylation/activity of NFAT (An important transcription factor in immune cells)
What 2 second messengers does PIP2 lipid hydrolysis generate?
How does IP3 increase cytosolic Ca2+?
By triggering Ca2+ release from intracellular stores