Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function

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,Chris Hague

doi:10.1074/jbc.m805251200

Jennifer L Wacker, David B Feller + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m805251200

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Abstract

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Highlights

Immunoprecipitation of wild type (WT) and L148S human G-protein-coupled receptor 54 (GPR54) (hGPR54)-GFP showed that both WT and mutant receptors were expressed at the expected molecular mass of ϳ70 kDa, demonstrating that the mutation does not interfere with protein expression (Fig. 1A)
Confocal analysis revealed that WT and L148S hGPR54-GFP cellular localization patterns were indistinguishable, suggesting that the L148S mutation does not cause trafficking or expression deficits of GPR54 (Fig. 1B)
Due to the overwhelming percentage of disease-causing G-protein-coupled receptor (GPCR) mutations that result in inefficient plasma membrane expression, we suspected that the L148S mutation of GPR54 might cause intracellular retention and explain the etiology of idiopathic hypogonadotropic hypogonadism (IHH)

Summary

EXPERIMENTAL PROCEDURES

Constructs—cDNA for human GPR54 (hGPR54) in pEFIN3 was kindly provided by Dr Marc Parmentier (University of Brussels). hGPR54 was amplified by PCR to add 5Ј EcoRI and 3Ј BamHI sites for subcloning into pcDNA3.1 containing an N-terminal FLAG or hemagglutinin (HA) epitope tag. 5Ј BamHI and 3Ј XhoI sites were added using PCR to subclone hGPR54 into pEGFP-N3 containing a C-terminal GFP epitope tag. Stable HEK293 cell lines expressing wild type (WT) or L148S hGPR54 were lysed overnight in buffer containing 1% digitonin. GFP Immunoprecipitation/Immunoblotting—Confluent 150-mm plates of HEK293 cells were washed with PBS, scraped, and centrifuged at 5,000 ϫ g at 4 °C for 5 min. For hGPR54 radioligand binding assays, samples were incubated with increasing concentrations of 125I-labeled KP-10 (PerkinElmer Life Sciences), a synthetic full agonist for GPR54, in the absence (total bound) or presence (nonspecific bound) of 0.5 ␮M unlabeled KP-10. Cells were incubated for 1 h at 4 °C, pelleted, washed three times in PBSϩ, resuspended in secondary antibody at 1 ␮g/ml (Invitrogen GAM Alexa Fluor௡), and incubated for 30 min at 4 °C. Co-immunoprecipitation of GPR54 and Gq Family Members— 150-mm plates of HEK293 cells were transiently transfected with TAP-WT or L148S hGPR54 and G␣q or G␣15/16. Cells were imaged using a Zeiss SP1 confocal microscope with a 100ϫ oil immersion objective

RESULTS

Protein identity

WT alone

DISCUSSION