Adrenergic receptors

Question 1

Features of b1-AR

Answer 1

isoprenaline > noradrenaline = adrenaline
↑ [cAMP] ->↑ PKA-> ↑ muscle contraction (fight or flight response)

Question 2

Features of b2-AR

Answer 2

isoprenaline > adrenaline&raquo_space; noradrenaline
↑ MAPK ↑ cPLA2-> ↑ muscle contraction

Question 3

Structures of Adrenaline, Noradrenaline and isoprenaline

Answer 3

SEE PHOTO ON PHONE

Question 4

Features of alpha adrenergic receptors

Answer 4

Alpha 1 – activation of these increases things like PLC, IP3 and DAG – again linked to muscle contraction

Alpha 2 – downregulation of cAMP levels

Question 5

β−AR was the first GPCR to be …

Answer 5

characterised by radioligand binding studies

cloned and expressed (Avian β1 and Hamster β2; Human isolated by low stringency screening of cDNA libraries)

structurally determined by crystallography (Rasmussen 2007)

Question 6

What is the model system for GPCR pharmacology, singling and regulation?

Answer 6

β−AR:

> 800 GPCRs have been revealed since the sequencing of the human genome

~15% of drug targets

e.g., AR agonists and antagonists used for treatment of asthma and cardiac arrythmias, respectively

Question 7

Primary structure of b-AR

Answer 7

7 transmembrane domains

Glycosylation sites – extracellular face

Intracellular disulphide bond

Large intracellular loops

Question 8

What about the structure of noradrenaline gives it specificity and direction

Answer 8

Hydroxyl groups

Question 9

Conserved negatively charged AAs on b-adrenergic receptors

Answer 9

Asp 113 in TM3 (even a change to Asn (abolishing negative charge) gave no binding)

Asp79 and Asp318 (showed decreased agonist binding but less of an effect on antagonist binding)

Question 10

Explain experimental techniques that allow discovery of info on how drugs bind to b-adrenergic receptors

Answer 10

Site directed mutagenesis (Isoyaga 1998)
and
Looked at selectivity of subtypes using chimeric proteins

Question 11

Explain how site directed mutagenesis gave drug binding info for the b-adrenergic recpeptors

Answer 11

Isoyaga 1998

Going through all the different hamster, human, rat, bovine adrenergic receptors, we see serines that are highly conserved - Ser204 + Ser207 (also Ser203 in TM5?)

mutation studies undergone to see effect on binding

Results:

• very low Kds (high affinity) when you have hydrogen bond donors and the two hydroxyl groups of the drug
• knock off the hyroxyl grouos from drug – increase in Kd (reduced affinity)
• abolish OHs in the receptor binding site (replace Ser with Ala) - increase in Kd (reduced affinity)

Question 12

Explain how the selectivity of the b-adrenergic subtypes was assessed using chimeric proteins

Answer 12

Taken WT b2 adrenergic receptor and WT b1 adrenergic receptor - which have slightly different pharmacologies ,take parts from both and reconstitute to get functional receptor

Make a series of different clones by:
- Select various transmembrane domain and start to couple them together e.g taken TM domain 2-7, replaced first TM domain with one from the B1-AR, e.g. again second TM domain replaced (Can also be done other way round)

Salmeterol a B2-AR agonist
125*I-CYP – antagonist

look at inhibition by Salmeterol

Look at TM domain 3 and to a certain extext 7 – start to regain the pharmacology of the B2 receptor when you look at the CH3 and CH7 constructs
These are the domains that are giving you the selectivity in this case
(Similar experiments have been done with the muscarinic Ach recptor)

Question 13

Explain the model for b-adrenergic receptor drug binding by the use of Chimeric protein studies and site directed mutagenesis

Answer 13

TMD 3 we have Asp113 which is making important dynamic interactions with the nitrogen

Ring structure – two OH groups form important H bonds with Serine 207 and Serine 205 of TMD5

Again we are seeing this agonist binding site is located within the TMD and is bridging netween TMD 3 and 5

Question 14

What do other studies suggest about Ser203s role in b-adrenergic drug binding

Answer 14

ser203 also binds to the same OH as ser204

Question 15

What confers b2-AR specificity

Answer 15

Tyr308 in TM7 (found by mutagenesis studies converting Tyr to Ala)

Question 16

Why is it important to understamd subtype specificity in b-ARs?

Answer 16

Allows development of therapeutics that only target a particular class of receptor (have selectivity for one and not another) and thefore could be found in a particular part of the body e.g. brain

this minimises off target effects

Question 17

Explain how some receptors have intrinsic activity

Answer 17

B1AR and B2AR – even in absence of agonist they stimulate cAMP production

We see a concentration dependent effect, increase Rs, increase cAMP levels – must be activating pathways within the cell

Question 18

Out of the b-ARs, which show a higher basal level

Answer 18

b2AR (5 fold higher)

Question 19

What human polymorphism in b-ARs is associated with heart disease

Answer 19

T164I human polymorphism reduces the basal activity of ß2AR to that of ß1AR

Question 20

Explain the activity equilibrium that exists in b-ARs

Answer 20

receptors exist in two conformational states

equilibrium between a high affinity (R* state) and a low affinity (R state) - the two coexist in equilibrium

Question 21

Explain what a graph for the competative binding of iosprenaline with 125I-CYP would look like for b-ARs, what this looks like it shows, and what it actually shows

Answer 21

y-axis 125I-CYP bound
x-axis log[isoprenaline] nM

waved curve going doenwards
peaks of these two waves show:
Two different binding modes:
Lower concentrations -High affinity – Kd 10-9M
Higher concentrations – Lower affinity – Kd 10-6M

Looks like: 2 binding sites (with different Kds)

Reality: receptors exist in two conformational states (R and R*)

Question 22

What is a good model for ligand binding to b-ARs

Answer 22

Basal rate at rest - R more than R*

Full agonists incrase R* population

Antagonists bind equally to the two states

Partial agonists – bind more the R* state to the R state

Inverse agonists – bind to R state and suppressing activity of active R conformation

Question 23

Explain experimental methods for the discovery of the sequences responsible for receptor binding of b-ARs

Answer 23

Strader 1987

Deletion mutatnt between 222-229 – no coupling, only 1 binding site

Deletion from 229-258 (intracellular loop 3) – coupling and 2 binding sites

Experiments with αAR/ßAR or ß1/2 (hybrid receptors)

Use i3 loop from ß2 in α2 receptor = ↑cAMP - replacing the selectivity

Swapping ß1/2 i3 loops - changes amount of cAMP produced, no coupling and single binding site

Overall:

Deletion of 222-229 and 258-272 decrease coupling to Gs

These are regions which are confiring selectivity of the particular G protein

Question 24

Explain the experimental techniques involving the DRY motif of b-ARs to determine equilibrium of the R and R* states

Answer 24

Ballesteros 2001

at the end of the third TMD - DRY motif
Important for determining equilibrium between active and inactive receptors
- Take DRY motif and look at level of basal coupling to the G proteins
- DRY motif intact – basal level of G protein activation is relativly low - Stabilizes the (R) state of the GPCR
- Disruption of DRY motif (by mutating residues) enhances basal activity - activation of receptors even in the absence of an agonist

Question 25

Stratagies for GPCR crystallisation

Answer 25

Very mobile makes it difficult, overcome by trying to enhance the region of the protein which is available for forming crystal crystal contacts (stabalise structure anbd increase surface for crystallisation)

This allows a crystal lattice to form that is needed for X-ray crystallography

Question 26

How are stratigies for GPCR crystalisation done

Answer 26

1. Clones lysozyme (which readily crystalises) into sequence of GPCR, Make use of low SA of lysozytme to promote contact between different proteins - drives crystal formation
2. Conformationally specific antobodies e.g. Fab fragments that bind to regions of protein, these crystralise readily

Question 27

Downside of the GPCR crystallisation techniques

Answer 27

High resolution structures close to where antibody is bound, but on opposite face – there’s a lot of conformational disorder, which is reflected in a lot lower resolution

In protein databases – may find large regions of beta sheet due to where people have cloned in things like Lysozyme of Ab fragments

Question 28

Structure of R state b2-AR

Answer 28

crystalised with lysoszyme

• Classical helical bundle
• 4 +3 helices
• Lysozyme replaces large flexible loop 3
• Small extracellular helix (Extracellular helix 2 – ECL2)

What do we see?
At the top – agonist binding site/binding site entrance
Between helices 3,4 and 5 – interface – Cavity in centre of bundle

Question 29

What is the inverse agonist of b2-ARs

Answer 29

Carazolol

Question 30

Explain binding of the inverse agionist Carazolol to the b2-adrenergic receptor

Answer 30

• Polar Interactions with ligand (Asp113, Ser203, Asn312, Tyr316)
• Hydrophobic interactions with ligand (Val114, Phe 290, Phe193)
  Therefor ligand binding is driven by polar and hydrophobic interactions

Location of binding site similar to muscarinic receptor – cavity exposed to the extracellular surface, so that ligand can enter binding site

Question 31

Agonists to the b2-AR binding site

Answer 31

Adrenaline
HBI
BI167107

Question 32

Properties of Agonists in the b2-AR binding site

Answer 32

Ring 2013
this case used an antibody fragment called a nanobody to get crystallisation
- All 3 bind with a very similar binding motif
- residues important in binding - Asn293, Ser203, Ser207, all form H bonds with ligands in binding site
- Phe (F193) coming down and making interactions with ligand

Question 33

Explain how the crystal structure of the GPCR coupled to Gs protein was obtained

Answer 33

Rasmussen 2011

To obtain crystallisation at a high enough resolution, used a T4 lysozyme (introdueced between TM4/5) and a nanobody (nb35)

Done in the precence of BI-167107

GDP/GTP was removed by Apyrase to ensure stability

crystals diffract to 2.9A resolution

Question 34

Explain the results seen when the active (R*) crystal structure of GPCR (couples tp Gs) was obtained

Answer 34

Rasmussen 2011

• Rearrangement of 7TMD scaffold
• 14A movemonet of TM6
• Outward movmement + extension of TM5 by 7 residues
• Outward movement of TM5&6 creates cavity in centre of helical body
• ICL2 : converted from helical to disordered - disrupting the ionic interactions that stabalise the inactive conformation

Large cavity that has been created is where g protein binds

Question 35

Explain the binding/exiting of the G-protein (Gs) to the GPCR

Answer 35

Rasmussen 2011

α5 (C terminal helix of G protein) forms hydrophobic interactions with GPCR core

As α5 exits the receptor a number of polar/ionic interactions are made with TM3/5 - some are involved in breaking ionic lock (IL2 bridges from α5 to the conserved DRY motif):

D(E)RY Motif Disrupted

Asp130/Arg131 make no salt bridge to Glu268 to lock receptor in the inactive conformation.

Arg131 forms essential interaction with Tyr391 of Gαs α5 helix

Question 36

Explain G protein activation of the GPCR

Answer 36

Rasmussen 2011

Large reorientation of domains in G protein

Large displacement of α5 helix

β6/α5 : involved in guanine ring binding displaced see below

At other end of a5 helix (not side binding to CPCR) is the nucleotide binding site – so binding of `G protein reduces affinity for GDP and GDP will disassociate

Now GTP binds to binding site - makes favourable interactions with different regions of the G protein and activates it by - P-Loop (β1-α1) displaced (breaks interface between the alpha and the beta, gamma subunits)

Question 37

From the information obtained in Rasmussens crystallisation of the active GPCR-Gas form, give the overall mechanism of G protein binding and activation

Answer 37

Receptor binds G protein with GDP bound

C-terminal helix (a5) gets pulled away from guanine group, at same time interaction of N terminal domain

Release of GDP and binding of GTP

Causes disruption of interface between G protein and receptor

Disassociation of beta, gamma subunits

Release of active complex from receptor complex