GPCR Structural Motifs Flashcards
Molecular basis for Family A GPCR activation
- role of the 2nd extracellular loop
- the vestibule: role in ligand entry and exit for alpha-branch family A GPCRs
- the movement of the helices
- the role of the DRY motif
Role of 2nd extracellular loop of family A GPCRs
- ECL2 of family A GPCRs is the largest and most diverse fo the extracellular loops
- ECL2 contributes to ligand:
- recognition
- binding
- selectivity
- allosteric modulation
- activation of GPCRs
- computational studies of beta-2 adrenoceptor suggest that small molecule ligands “pre-bind” to the receptor in the area around the ECL2 before travelling down to the orthosteric binding site and that a ECL2 salt bridge is stabilised by F193 (ECL2) regulating orthosteric ligand binding
- Phe193 (ECL2) and Tyr308 must separate for ligands to move from the extracellular region to the binding pocket
Role of vestibule in ligand exit for alpha-branch family A GPCRs
COME BACK TO THIS
i’m guessing it slows ligand unbinding but need to check lecture recordings
Aromatic lid - alpha-branch family A GPCRs
- aromatic live above orthosteric site
- binding of a ligand to alpha-branch family A GPCRs helps close the lid over the entrance to the binding pocket, thereby restraining the ligand within the pocket
How does ligand binding result in changes in the intracellular side of the receptor?
- ligand binding
- conformational changes
- cellular response
How does GPCR activation occur?
Hypothesis: GPCR activation occurs via movement of the transmembrane helices
- results suggest that TM6 moves closer to TM5 and away from TM3 by either a rotation or a lateral movement of TM6
- the movement of transmembrane helix 6 allows for the insertion of alpha-5 helix of G protein
- the g protein alpha-5 helix is the primary site of receptor-G protein interactions
E/DRY motif for Family A
- DRY motif is found at the bottom of helix 3/start of intracellular loop 2
- hydrogen bond network stabilise family A GPCRs
- different residues stabilise the receptor in the inactive state compared to the active state
How does ligand binding result in conformational changes at the intracellular surface of the receptor?
Ligand binding into the orthosteric site induces conformational changes that are transmitted to the intracellular loops and tail.
Conformational changes include:
- Transmembrane helix 6 (TM6) moving closer to TM5 and away from TM3
- Leads to the breaking of hydrogen bonds between the intracellular ends of TM3 and TM6. The residues involved in this hydrogen bonding network are R3.50, D3.49 and E6.30 (these were the residues that stabilised the inactive state)
- A tyrosine residue in intracellular loop 2 (ICL2) and a tyrosine in TM5 are also involved in the hydrogen bonding network between TM3 and TM6
- The movement of TM6 then allows Y7.53 of the conserved NPxxY motif to move into the space previously occupied by TM6 in the inactive state
RAMPs
- Receptor activity-modifying proteins (RAMPs) are membrane-spanning accessory proteins
- RAMPs can alter trafficking, signalling and pharmacology of GPCRs
- Comprise of a single transmembrane spanning domain with an extracellular N-terminal domain of ~90-100 amino acids and a short intracellular C-terminal domain of ~9 amino acids
- RAMP1 promotes stabilisation of the ECD of CLR (calcitonin-like receptor, a Family B GCPR), which alters how the ligand interacts with the receptor core
Family B - RAMPs alter receptor pharmacology: 2 receptors become 7
- Calcitonin (CT) receptor (CTR) and calcitonin-like receptor (CLR) interact with multiple peptide ligands
- Interaction of three different RAMPs with CLR or CTR gives rise to seven receptors which have differing ligand selectivity profiles
i. e. depending on which RAMP interacts with the receptor different agonists/antagonists will be more likely to bind
RAMPs and Family B GPCR Expression and Trafficking
- RAMPs have a chaperone role in receptor glycosylation and expression
- The calcitonin like receptor (CRLR) needs heterodimerisation with a RAMP for efficient transport to the cell surface
RAMPs and Family C GPCR Expression
RAMPs have a chaperone role in receptor glycosylation and expression
- During synthesis the CaS receptor (CaSR) polypeptide, together with the ribosome, is targeted to the rough ER
- After synthesis, the receptor is inserted into the ER membrane
- In the ER, CaS receptor monomers assemble as homodimers, which are retained and are core glycosylated
- Association with RAMP1 or RAMP3 allows the CaSRs to escape the ER retention and reach the Golgi apparatus where they are terminally glycosylated before being inserted into the plasma membrane (PM)
RAMPs and Family B GPCR Signalling
RAMPs can change the ability of a receptor to couple to different signalling pathways
- a receptor alone may preferentially signal via cAMP pathway whereas a receptor plus RAMP2 may preferentially signal via PI hydrolysis
Possible roles of GPCR dimerisation
- receptor maturation and correct transport of GPCRs from the endoplasmic reticulum to the cell surface
- dynamic regulation of dimers by ligand binding
- changes to ligand selectivity
- modulation of signalling or G-protein selectivity
Family C function as dimers
- Cis activation - agonist binding in one VFT can activate the 7TM of the same receptor
- Trans activation - agonist binding in one VFT activates the 7TM of the other receptor
- The GABAB (heterodimer) receptor only activates via trans-activation, with agonist binding to the GABAB1 VFT leading to the activation of the GABAB2 7TM
- The mGluRs (homodimers) can be activated by both cis and trans-activation
