Receptors Flashcards
What percentage of drugs act on GPCRs?
50%
Generally how do GPCRs work
- Integral membrane proteins interact with some ligands
- conformational change transferred to G-protein and produces signalling cascade
Where are beta 2 adrenergic receptors mostly found?
In smooth muscle
What hormones do beta2 adrenergic receptors respond to?
Adrenaline and noradrenaline
When beta2 adrenergic receptors are activated by an agonist what are they used to treat?
Asthma and premature labour
When beta2 adrenergic receptors are inactivated by an anagonist what are they used to treat?
Hypertension or cardiac arrythmia
Why was it so difficult to get a structure of beta2 adrenergic receptors?
- Low concentration in tissue
- Unstable once purified
- Flexible and dynamic
Why was T4 lysozyme added to beta2 adrenergic receptors?
- Likes to crystallise
- Enhances ability of a protein to crystallise
Where was lysozyme put into beta2 adrenergic receptors?
It replaced the 3rd intracellular loop
What was used to recombinantly express beta2 adrenergic receptors?
Insect cells
Which part of beta2 adrenergic receptors was deleted to aid crystallisation?
48 C-terminal amino acids
What 3 methods were used to help get a structure of beta2 adrenergic receptors?
- Mutation
- Addition of lysozyme
- Addition of antibody
How were beta2-adrenergic receptors purified?
Using antibodies and ligand affinity columns
What were beta2-adrenergic receptors crystallised in the prescence of?
Carazolol
Why is carazolol used to help crystallise beta2 adrenergic receptors?
It is a partial inverse agonist and so forces the protein into an inactive state
How is lysozyme attached to the beta2 adrenergic receptor?
Via the 3rd intracellular loop
How does the beta2 adrenergic receptor bind to ligands?
- Forms a network of H bonds to side chains
- Multiple hydrophobic contacts
Why does lysozyme fusion aid crystallisation?
Specific contacts with lysozyme reduce conformational flexibility and help to stabilise beta2 adrenergic receptor
Why was it difficult to crystallise the full GPCR-Gs complex?
- Movement of the Gαs subunit (it is inherently flexible)
- Large detergent micelle around the protein means there is no avaliable surface for crystal lattice structures
What is a nanobody?
An antibody without a light chain
How did nanobodies aid the crystallisation of Beta2-adrenergic receptors?
They locked the G-protein in conformation
What are the major differences between the active and inactive state of beta2 adrenergic receptors?
- Transmembrane helice 6 moves outward by 14Å
- Small extension of TM helix 5 becomes more structured
- Intracellular loop2 changes from an extended loop to an α-helix
