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Neuroscience > GPCRs > Flashcards

GPCRs Flashcards

1
Q

Location of 11-Cis Retinal on Rhodopsin

A

Lys296 on TM7

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2
Q

Asp79 and Asp113 on TM3

A

Mouse fibroblast L cell line

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

Asp79
mutation appeared more critical for efficacy

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3
Q

Ser204 and Ser 207 on TM5

A

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

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4
Q

Ser203

A

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
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5
Q

Asn293

A 
Asn293Leu
Faces binding site when applied to bacteriorhodopsin structure
Stereospecific to R- isomer
Greater for full vs. partial agonists
Did not affect antagonists
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6
Q

Purpose of Fabs

A

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

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7
Q

Purpose of T4 Lysozymes

A

Inserted into EC loops to increase crystal contact and resolution

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8
Q

Active rhodopsin structure

A

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

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9
Q

Nb80 for active state B2AR

A

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

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10
Q

Structural change in B2AR activation

A

Outward displacement of TM5 and 6

Inward movement of TM3 and 7

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11
Q

Nb6B9

A

10x affinity of Nb80 (allowed greater resolution)

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

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12
Q

Actual interactions of B2AR after active state resolution

A

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

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13
Q

Ser204 and Asn293 actual role

A

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

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14
Q

Ser207

A

Side-chain rotation in Pro288 acts as a hinge region

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15
Q

Adenosine Receptor (EC binding)

A

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

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16
Q

Neurotensin receptor (EC binding)

A

8-13 residues critical. Very superifcial

17
Q

PAR-1

A

Cleavage of N-terminus by proteinase produces new n terminus

Synthetic agonist S42FLLRN made of critical residues binds receptor

18
Q

Lipid Activated receptors

A

Entrance blocked by EC2/ TM3

19
Q

Family B GPCRs examples

A

PTH
Glucagon
Calcitonin
GLP-1R

20
Q

Family B activation

A

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

21
Q

NAM sites for Family B receptors

A

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

22
Q

Family C GPCRs examples

A

mGluRs
PT Calcium Receptor
GABAr

23
Q

Family C receptor binding

A

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

24
Q

Samama paper- key points

A

Mutated B2AR with 4AA changes in C terminal

COS-7 transfection underrepresented compared to WT

25
Q

Key Data from Samama paper

A

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

26
Q

Why did TCM need to be extended

A

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

27
Q

Yao key points

A

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

28
Q

Yao Key data

A

Agonist/antagonist/inverse agonist additon

Uncoupled Gs similar to unliganded receptor- similar activation levels

Preincubation- agonist, antagonist, inverse antagonists
- no true antagonists, propanolol closest

29
Q

Chen key points

A

Variable cDNA expression in hCTR2/NPY1/CXCR-1 receptors

Measured melanin dispersion as a function of Adenylyl Cyclase activity

30
Q

Chen key data

A

Maximum constitutive activity 60% cAMP activity of full activity

Agonist addition at 4 less than constitutive at 16
AC512 inverse agonist addition

Neuroscience (17 decks)

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