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GPR54 the Second Intracellular Loop Class A G-protein-coupled - - PowerPoint PPT Presentation
GPR54 the Second Intracellular Loop Class A G-protein-coupled - - PowerPoint PPT Presentation
GPR54 the Second Intracellular Loop Class A G-protein-coupled Receptor Jennifer L. Wacker, David B. Feller, Xiao-Bo Tang, Mia C. DeFino, Yuree Namkung, John S. Lyssand, Andrew J. Mhyre, Xu Tan, Jill B. Jensen, and Chris Hague Journal of
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G-protein Signaling Transduction
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Gα effecters G β γ effecters G β G γ G α GTP GDP
▲ Extracellular
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GPR GPR54
A member of Class A GPCR A receptor of kisspeptin Modulates gonadotropin- releasing hormone (GnRH) secretion
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How the reproductive system work?
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Hypothalamic-pituitary-gonadal Axis
5 gonads Pituitary Hypothalamus
Gonadotropin-releasing hormone Luteinzing hormone Follicle-stimulating hormone Sexual steroid secretion
Idiopathic hypogonadotropic hypogonadism
GPR54*
G α G β G γ
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IHH IHH
Results from disorder of GnRH secretion Failure to initiate puberty, immature reproductive
- rgans, infertility
A mutation in GPR54 (L148S) in previous study causes IHH
6 gonads Pituitary Hypothalamus
GnRH LH, FSH Sexual steroid secretion
GPR54 (L148S)
G α G β G γ
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GPCR54 Structure
7TM domain The second intracellular loop IL2-10 Leucine148 → serine
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▲ Extracellular ▼ Intracellular
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Why L148S Cause IHH?
G α G β G γ
▼ Intracellular
GDP G α G β G γ GDP GTP
▲ Extracellular
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Results of Immunoprecipitation and Confocal Microscopy
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L148S expression and localization are equivalent to WT GPR54.
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Results of Ligand-binding Assay
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GDP G α G β G γ
L148S does not abolish ligand-binding property of GPR54.
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Dose L148S Abolish the Functional Coupling with Gα?
G β G γ
PhospholipaseC
PI DAG G α GTP
Phosphoinositol (PI) hydrolysis
IP3
▲ Extracellular
GDP
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Results of PI Hydrolysis Assay
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KP-54: The natural full-length ligand KP-10: An artificial ligand
G α G β G γ GTP
L148S interrupts the functional coupling of GPR54
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Two possible mechanisms by which L148S Interrupts the Functional Coupling
GDP G α G β G γ G α G β G γ GDP GTP
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Results of Co-immunoprecipitation
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G α G β G γ GDP
L148S does not abolish Gα binding
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FR FRET
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Fluorescence resonance energy transfer FRET occurs between the Gαq-CFP and Gβγ-YFP As GPR54 is activated, FRER signal decrease
G β G γ
YFP
G α
CFP
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Results of FRET Assay
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G α G β G γ GDP GTP
L148S is unable to activate Gα to cause it dissociates from Gβγ.
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Summary 1
L148S GPR54 remains normal expression, localization, and kisspeptin-binding properties. L148S GPR54 can bind to Gα subunit as wild-type GPR54 does. L148S lose the ability to activate Gα.
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GPR54 (L148S)
G α G β G γ
Pituitary Hypothalamus
GnRH IHH
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GPCR54 Structure
7TM domain The second intracellular loop Leucine148 → serine
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▲ Extracellular ▼ Intracellular
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IL2-10 Is Highly Conserved in Class A GPCRs
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S Adrenergic receptor alpha 1A
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Structural Modeling of GPR54/Gαq complex
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GPR54/β2-AR Gαq
IL2 and its conserved residue are critical determinants of hydrophobic interaction between G α for Class A GPCRs.
Limitation of GPCR/Gα co-crystal
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Results of Mutation Analysis
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The presence of hydrophilic, acidic, basic amino acids inhibits functional coupling
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Summary 2
The sequence analysis reveals conserved hydropholic residues amount Class A GPCRs. These conserved residues are required for GPCR functional coupling. Structural modeling shows the hyprophobic interface created by IL2 is critical for interaction with Gα.
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Leu148 of GPR54 Is a Critical G-protein Interaction Site
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L148S causes GPR54 lose activity toward Gα. L148S affects the interaction between GPR54 and downstream molecules. IL2 region and IL2-10 residue are essential for Class A GPCRs.
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Than anks f for Att tten entions & & Mer erry X y X’ ’ Mas!
Special thanks to Pro. Yang for helping me a lot.
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