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 Biological Chemistry Papers in Press, published online ahead of print September 4, 2008 1

GPC GPCR ▲ Extracellular G-protein-coupled receptor Seven transmembrane domain (7TM) Controls many critical signal transduction pathways G α subunit Class A to Class F G βγ subunit 2

G-protein Signaling Transduction ▲ Extracellular G γ G α G α effecters G β GTP GDP G β γ effecters 3

GPR GPR54 A member of Class A GPCR A receptor of kisspeptin Modulates gonadotropin- releasing hormone (GnRH) secretion How the reproductive system work? 4

Hypothalamic-pituitary-gonadal Axis Hypothalamus GPR54* Gonadotropin-releasing hormone G γ G α G β Pituitary Idiopathic hypogonadotropic Luteinzing hormone hypogonadism Follicle-stimulating hormone Sexual steroid gonads secretion 5

IHH IHH Results from disorder of GnRH secretion Hypothalamus GPR54 (L148S) Failure to initiate puberty, immature reproductive organs, infertility GnRH G γ G α G β Pituitary A mutation in GPR54 (L148S) in previous study causes IHH LH, FSH Sexual steroid gonads secretion 6

GPCR54 Structure ▲ Extracellular 7TM domain The second intracellular loop IL2-10 Leucine 148 → serine ▼ Intracellular 7

Why L148S Cause IHH? ▲ Extracellular G γ G γ G α G β G α G β GTP GDP ▼ Intracellular GDP 8

Results of Immunoprecipitation and Confocal Microscopy L148S expression and localization are equivalent to WT GPR54. 9

Results of Ligand-binding Assay G γ G α G β GDP L148S does not abolish ligand-binding property of GPR54. 10

Dose L148S Abolish the Functional Coupling with G α ? Phosphoinositol (PI) hydrolysis ▲ Extracellular PI DAG G γ G α G β GTP GDP PhospholipaseC IP3 11

Results of PI Hydrolysis Assay KP-54: The natural full-length ligand KP-10: An artificial ligand G γ G α G β GTP L148S interrupts the functional coupling of GPR54 12

Two possible mechanisms by which L148S Interrupts the Functional Coupling G γ G γ G α G β G α G β GTP GDP GDP 13

Results of Co-immunoprecipitation L148S does not abolish G α binding G γ G α G β GDP 14

FR FRET Fluorescence resonance energy transfer G γ FRET occurs between the G α q -CFP G α G β and G βγ -YFP CFP YFP As GPR54 is activated, FRER signal decrease 15

Results of FRET Assay L148S is unable to activate G α to G γ G α G β cause it dissociates from G βγ . GDP GTP 16

Summary 1 Hypothalamus GPR54 (L148S) GnRH G γ IHH G α G β Pituitary L148S GPR54 remains normal expression, localization, and kisspeptin-binding properties. L148S GPR54 can bind to G α subunit as wild-type GPR54 does. L148S lose the ability to activate G α . 17

GPCR54 Structure ▲ Extracellular 7TM domain The second intracellular loop Leucine 148 → serine ▼ Intracellular 18

IL2-10 Is Highly Conserved in Class A GPCRs Adrenergic receptor alpha 1A S 19

Structural Modeling of GPR54/G α q complex Limitation of GPCR/G α co-crystal GPR54/ β 2 -AR G α q IL2 and its conserved residue are critical determinants of hydrophobic interaction between G α for Class A GPCRs. 20

Results of Mutation Analysis The presence of hydrophilic, acidic, basic amino acids inhibits functional coupling 21

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 G α . 22

Leu148 of GPR54 Is a Critical G-protein Interaction Site L148S affects the interaction between GPR54 and downstream molecules. L148S causes GPR54 lose activity toward G α . IL2 region and IL2-10 residue are essential for Class A GPCRs. 23

Than anks f for Att tten entions & & Mer erry X y X’ ’ Mas! Special thanks to Pro. Yang for helping me a lot. 24

FRET 436 nm FRET CFP* YFP CFP* YFP* 535 nm 480 nm detector The cyan fluorescent protein (CF CFP)- yellow fluorescent protein (YF YFP) pair is the most popular FRET pairs in biological use. 25