L11 - GPCR ligand binding and activation

Question 1

how many amino acids are there

Answer 1

20

Question 2

4 residue interactions

Answer 2

hydrophobic, electrostatic, hydrogen bonds, disulphide bonds

Question 3

transmembrane alpha helices are _____

Answer 3

amphipathic/amphiphillic

Question 4

which amino acid breaks alpha helices

Answer 4

proline

Question 5

which amino acids can be phosphorylated

Answer 5

Serine, tyrosine and threonine

Question 6

which amino acid forms disulphide bonds

Answer 6

cysteines

Question 7

describe orthosteric binding by the class rhodopsin

Answer 7

orthosteric binding site is in the upper transmembrane domain and sometimes involves the extracellular domain if the ligand is larger

Question 8

describe orthostreic binding by the class secretin

Answer 8

recognised by both transmembrane domain and extracellular domain

Question 9

describe orthosteric binding by the class glutamate

Answer 9

Binding occurs in the extracellular domain that contains the venus flytrap domain

Question 10

describe orthosteric binding by the class adhesion

Answer 10

Little is known about binding at these receptors however, they contain large ECDs

Question 11

describe orthosteric binding by the class frizzled

Answer 11

both smoothened and frizzled receptors possess an ECD that contains a cysteine rich domain and a ECD-linker domain. WNT binds at the CRD of FRZ receptors

Question 12

what is the function of allosteric interaction networks

Answer 12

They enable the relay of binding events through the transmembrane domain to the cytosolic portion of the receptor

Question 13

what is the common feature of agonist activation amongst almost all GPCRs

Answer 13

the outward movement of TM6 opens up the cystolic portion of the receptor to allow for the binding of G proteins

Question 14

movement of Class A TM6 domain

Answer 14

35º

Question 15

movement of Class B domain

Answer 15

50º

Question 16

movement of Class F domain

Answer 16

13º

Question 17

Due to the highly diverse ligand binding pockets and conformational changes in Class A GPCRs binding how is the diversity mediated to produce consistent receptor response

Answer 17

The contain several conserved motifs resulting in common structural rearrangements of residue contacts between TMs 3,6 and 7

Question 18

what is an allosteric interaction network

Answer 18

amino acid residue that are composed of various conserved motifs and residues (microswitches)

Question 19

what are the conserved motifs in Class A

Answer 19

CWxP, PIF, DRY (D(E)RY) and the ionic lock, Na+ binding site, NPxxY

Question 20

what is a conserved structural residues

Answer 20

they stabilise a receptor as it changes confirmations

Question 21

what are examples of conserved structural residues in Class A GPCRs

Answer 21

Cysteines in ECD, Prolines in TM5, 6 and 7 - they allow for relatively large
structural changes at the cytoplasmic
side of the receptor and minimal change at the binding pocket

Question 22

what are the characteristics of the Beta 2 adrenoreceptor

Answer 22

• well characterised GPCR
• Gs coupled receptor
• binds endogenous monoamines (adrenaline)
• causes relaxation of Visceral SM, bronchodilation, vasodilation, hepatic glycogenolysis, muscle temor
• one major drug target of asthma treatment (salbutamol) and hypertension (propranolol)

Question 23

Important residues for β2AR orthosteric agonist binding

Answer 23

D113 (aspartic acid) and N312 (Asparagine) are KEY for ALL B2adr ligand binding

S203 and S207 (Serine) form H
bonds with agonists

Various hydrophobic interactions are also important for stabilising or activating the receptor

Question 24

mechanism of action of B2AR activations

Answer 24

1. Adrenaline interactions with serines in TM5 causes rearrangement of various hydrophobic interactions (CWxP and PIF), moving the serines inward, creating a bulge at TM5 and
   contraction of the TM3-5-6 interface near the binding pocket.
   - Minimal conformation change at this
   stage.
2. Collapse of the Na+ pocket leads to denser repacking of various residues, initiating the movement of TM7 toward TM3 and rotation of the cytoplasmic end of TM6.
3. Rewiring of various contacts (NPxxY) strengthens the packing of TM3-TM7, while the packing of TM3-TM6 is further loosened with the outward movement of TM6.
4. These movements break the “ionic lock” (R in DRY with E in TM6), breaking the remaining contacts between TM3 and TM6 in the cytoplasmic end and driving the outward movement of TM6.
   - R interacts with the G protein
   - D(E) and Y form new contacts that
   stabilise the active state.
5. The cytoplasmic side of the receptor opens up to create a binding pocket for the Gα subunit.
   - This pocket is lined by residues from
   TM3, TM5 and TM6 and stabilised by
   water

Question 25

how do ligands bind to class b receptors

Answer 25

These receptors undergo two step binding 1. Rapid interaction between N-terminal extracellular domain of the receptor and the C-terminal of the peptide 2. slower and complex step that involves the N-terminal of the peptide into the transmembrane domain

Question 26

how is the inactive state of class b receptors is maintained

Answer 26

conserved allosteric interactions e.g polar core maintain inactivity - the ECD and the TM receptor core may also be involved.

Question 27

What changes occur upon class b receptor activation

Answer 27

Marked kinking and an outward movement of the extracellular ends of TM6 and TM7 and an inward movement of the extracellular top of TM1

Question 28

what are the conserved motifs of class b receptors

Answer 28

Polar core stabilizes the inactive state, NPGQ motif stabilizes the receptor’s active state and induces a kink which enables a 20 Ångstrums outward movement of TM6 compared with 6–14 Ångstrums in class A GPCRs.

Question 29

ligand binding to class c receptors

Answer 29

In the inactive state, the TMD regions are apart from each other. Ligand binding causes a conformational shift of the VFT module from an open to closed state This alters the position and orientation of the TMDs within the receptor dimer that triggers G protein binding

Question 30

conserved residues in class c receptors

Answer 30

Ionic lock – stabilises the inactive state FxxCWxP motif – ‘trigger switch’ to couple ligand binding to TM6 rearrangement FxPKxY motif – at the intracellular end of TM7, stabilises the active conformation

Question 31

What is inverse agonism

Answer 31

If constitutive activity is present in a receptor, inverse agonists are able to reduce the functional response below basal levels, unlike neutral antagonists which can only reduce activity to basal levels

Question 32

Structural explanation fro inverse agonism and antagonism

Answer 32

The intracellular portion of a receptor upon binding of antagonists has minimal movement and even less o=for INV agonists - meaning cytoplasmic G proteins cannot bind to produce a response

Question 33

What is biased signalling

Answer 33

the concept that different system components stabilize different receptor conformations, leading to differential activation or inhibition of effectors.

Question 34

Structural explanation of biased signalling

Answer 34

There is evidence from some GPCR studies that: Gαs coupling requires/causes a larger intracellular cleft and greater outward movement of TM6, relative to Gαi and Gαq β-arrestin-biased coupling requires/causes movement of TM7 rather than TM6.

Question 35

GPCR allosterism

Answer 35

whereby binding of a ligand other than at the orthosteric site influences receptor function, either by direct modulation of receptor conformation or altering the receptor conformation induced by an orthosteric ligand