GPCRs

Question 1

Location of 11-Cis Retinal on Rhodopsin

Answer 1

Lys296 on TM7

Question 2

Asp79 and Asp113 on TM3

Answer 2

Mouse fibroblast L cell line

Asp113
Conservative (x300 decrease) and non-conservative (x8000) decreases in affinity

Asp79
mutation appeared more critical for efficacy

Question 3

Ser204 and Ser 207 on TM5

Answer 3

S204 and S207 determined to face binding site as only one helical turn apart

S120/165/203 mutations unsuccessful- did not produce immunoreactive protein

Antagonist activity unaffected- lacks a catechol group
TM5 interactions crucial for conformational change

Question 4

Ser203

Answer 4

HEK293 cells

Confirmed in all catecholamine receptors
MTSEA binding confirmed Ser203 faces the binding crevice, only binds Ser203

Ser203Cys

• Partial agonists greatest reduction in affinity
• Mutant HAL agonist lacking mOH greatest reduction
• does not affect HAL- without pOH

Question 5

Asn293

Answer 5

Asn293Leu
Faces binding site when applied to bacteriorhodopsin structure
Stereospecific to R- isomer
Greater for full vs. partial agonists
Did not affect antagonists

Question 6

Purpose of Fabs

Answer 6

Increase crystallisation by hiding hydrophobic regions e.g. cytoplasmic loops, increasing crystal contact

Question 7

Purpose of T4 Lysozymes

Answer 7

Inserted into EC loops to increase crystal contact and resolution

Question 8

Active rhodopsin structure

Answer 8

Opsin structure resolved
Determined TM3 and TM6 displacement
Opsin crystals soaked in retinal; 11-cis-retinal destroyed crystals by inducing conformational change

Question 9

Nb80 for active state B2AR

Answer 9

Similar to g-protein in WT and T4L stabilised receptors (mBBr reporter)
Interacts with TM3, 5-7

Question 10

Structural change in B2AR activation

Answer 10

Outward displacement of TM5 and 6

Inward movement of TM3 and 7

Question 11

Nb6B9

Answer 11

10x affinity of Nb80 (allowed greater resolution)

Tyr219-Tyr326 H bond mediated by water molecules in active state-similar to ionic lock

Question 12

Actual interactions of B2AR after active state resolution

Answer 12

BetaOH Asp113
Amine Group Asp113 and Tyr316 (TM7)
Catechol group Ser203=mOH and Ser207=pOH

Question 13

Ser204 and Asn293 actual role

Answer 13

Extended H-bond network with His296 (TM6) which interacts with catechol group in its entirety

Question 14

Ser207

Answer 14

Side-chain rotation in Pro288 acts as a hinge region

Question 15

Adenosine Receptor (EC binding)

Answer 15

Interacts with 4 EC residues, notably Phe168(Loop 2) and Glu169 (loop 3)
Na acts as NAM by binding residues in a water-filled pocket, collapses on activation

Question 16

Neurotensin receptor (EC binding)

Answer 16

8-13 residues critical. Very superifcial

Question 17

PAR-1

Answer 17

Cleavage of N-terminus by proteinase produces new n terminus

Synthetic agonist S42FLLRN made of critical residues binds receptor

Question 18

Lipid Activated receptors

Answer 18

Entrance blocked by EC2/ TM3

Question 19

Family B GPCRs examples

Answer 19

PTH
Glucagon
Calcitonin
GLP-1R

Question 20

Family B activation

Answer 20

2 stage activation with NTD and EC regions
G protein binds slightly deeper
TM6 movement important for ligand access

Question 21

NAM sites for Family B receptors

Answer 21

e.g. CP-376395 is a NAM to CRF receptor
Blocks transmission switch, preventing outward movement
Stabilises TM6=TM7 Hydrogen bonds

Question 22

Family C GPCRs examples

Answer 22

mGluRs
PT Calcium Receptor
GABAr

Question 23

Family C receptor binding

Answer 23

Dimeric structure, mediated by cholestrol in membrane- also promote recruitment to lipid rafts
LB1 and LB2 transmit conformational change to cysteine rich region

NAMs stabilised structural resolution of mGluR5
Stabilised TM3-TM6 salt bridge

Question 24

Samama paper- key points

Answer 24

Mutated B2AR with 4AA changes in C terminal

COS-7 transfection underrepresented compared to WT

Question 25

Key Data from Samama paper

Answer 25

Basal cAMP activity 3x higher in mutants despite under representation Weak agonists gain efficacy Medium agonists become full agonists Propanolol neutral agonist Uncoupled state mutant higher affinity than WT

Question 26

Why did TCM need to be extended

Answer 26

Uncoupled state (GppNHp) mutant higher affinity than WT Not increased M (mutants retain high affinity for full agonists in G protein absence) Not increased K (does not explain increased affinity of partial agonists at mutants

Question 27

Yao key points

Answer 27

Minimal cysteine B2AR mBBr attached to Cys265 GTPyS added preliminarily- prove functional receptor

Question 28

Yao Key data

Answer 28

Agonist/antagonist/inverse agonist additon Uncoupled Gs similar to unliganded receptor- similar activation levels Preincubation- agonist, antagonist, inverse antagonists - no true antagonists, propanolol closest

Question 29

Chen key points

Answer 29

Variable cDNA expression in hCTR2/NPY1/CXCR-1 receptors | Measured melanin dispersion as a function of Adenylyl Cyclase activity

Question 30

Chen key data

Answer 30

Maximum constitutive activity 60% cAMP activity of full activity Agonist addition at 4 less than constitutive at 16 AC512 inverse agonist addition