GPCRs Flashcards
Location of 11-Cis Retinal on Rhodopsin
Lys296 on TM7
Asp79 and Asp113 on TM3
Mouse fibroblast L cell line
Asp113
Conservative (x300 decrease) and non-conservative (x8000) decreases in affinity
Asp79
mutation appeared more critical for efficacy
Ser204 and Ser 207 on TM5
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
Ser203
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
Asn293
Asn293Leu Faces binding site when applied to bacteriorhodopsin structure Stereospecific to R- isomer Greater for full vs. partial agonists Did not affect antagonists
Purpose of Fabs
Increase crystallisation by hiding hydrophobic regions e.g. cytoplasmic loops, increasing crystal contact
Purpose of T4 Lysozymes
Inserted into EC loops to increase crystal contact and resolution
Active rhodopsin structure
Opsin structure resolved
Determined TM3 and TM6 displacement
Opsin crystals soaked in retinal; 11-cis-retinal destroyed crystals by inducing conformational change
Nb80 for active state B2AR
Similar to g-protein in WT and T4L stabilised receptors (mBBr reporter)
Interacts with TM3, 5-7
Structural change in B2AR activation
Outward displacement of TM5 and 6
Inward movement of TM3 and 7
Nb6B9
10x affinity of Nb80 (allowed greater resolution)
Tyr219-Tyr326 H bond mediated by water molecules in active state-similar to ionic lock
Actual interactions of B2AR after active state resolution
BetaOH Asp113
Amine Group Asp113 and Tyr316 (TM7)
Catechol group Ser203=mOH and Ser207=pOH
Ser204 and Asn293 actual role
Extended H-bond network with His296 (TM6) which interacts with catechol group in its entirety
Ser207
Side-chain rotation in Pro288 acts as a hinge region
Adenosine Receptor (EC binding)
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
Neurotensin receptor (EC binding)
8-13 residues critical. Very superifcial
PAR-1
Cleavage of N-terminus by proteinase produces new n terminus
Synthetic agonist S42FLLRN made of critical residues binds receptor
Lipid Activated receptors
Entrance blocked by EC2/ TM3
Family B GPCRs examples
PTH
Glucagon
Calcitonin
GLP-1R
Family B activation
2 stage activation with NTD and EC regions
G protein binds slightly deeper
TM6 movement important for ligand access
NAM sites for Family B receptors
e.g. CP-376395 is a NAM to CRF receptor
Blocks transmission switch, preventing outward movement
Stabilises TM6=TM7 Hydrogen bonds
Family C GPCRs examples
mGluRs
PT Calcium Receptor
GABAr
Family C receptor binding
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
Samama paper- key points
Mutated B2AR with 4AA changes in C terminal
COS-7 transfection underrepresented compared to WT
Key Data from Samama paper
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
Why did TCM need to be extended
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
Yao key points
Minimal cysteine B2AR
mBBr attached to Cys265
GTPyS added preliminarily- prove functional receptor
Yao Key data
Agonist/antagonist/inverse agonist additon
Uncoupled Gs similar to unliganded receptor- similar activation levels
Preincubation- agonist, antagonist, inverse antagonists
- no true antagonists, propanolol closest
Chen key points
Variable cDNA expression in hCTR2/NPY1/CXCR-1 receptors
Measured melanin dispersion as a function of Adenylyl Cyclase activity
Chen key data
Maximum constitutive activity 60% cAMP activity of full activity
Agonist addition at 4 less than constitutive at 16
AC512 inverse agonist addition
