Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function

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.

FIGURE 1.

The L148S mutation does not alter the expression, localization, or ligand-binding properties of hGPR54. A, immunoprecipitation of WT and L148S hGPR54-GFP. B, confocal microscopy showing the localization of WT and L148S hGPR54-GFP. C, cell surface expression levels of WT and L148S HA-hGPR54 measured using fluorescence-activated cell sorting analysis. D, saturation binding of ¹²⁵I-labeled KP-10 to membranes from WT and L148S hGPR54 expressing HEK293 cells. The results presented in A and B are representative of three experiments performed in duplicate, and C and D show the means ± S.E. of three or four experiments performed in triplicate. IP, immunoprecipitation; IB, immunoblot.

FIGURE 2.

Leucine 148 plays an important role in GPR54 functional coupling. A and B, the L148S mutation decreased the intrinsic activity as measured by PI hydrolysis in response to KP-54 (A) or KP-10 (B) of FLAG-hGPR54 cells. C and D, the L148S mutation also decreased the intrinsic activity, measured by PI hydrolysis in response to KP-54 (C) or KP-10 (D) of hGPR54-GFP cells. E, KP-10-mediated ERK activation was suppressed in L148S hGPR54-GFP cells. All results are expressed as the means ± S.E. of 3-5 experiments performed in triplicate. For A-D, results are normalized to the response of WT hGPR54 to 1 μm KP (maximal response). For the ERK assay (E), results are normalized to ERK activation in response to 1 nm epidermal growth factor.

FIGURE 3.

The highly conserved Leu¹⁴⁸ of GPR54 is a critical determinant of effective G-protein coupling for diverse Class A GPCRs. A, alignment of IL2 of a number of Class A GPCRs highlights the conservation of proline and leucine residues (^*) downstream of the DRY motif that initiates IL2. B, [³H]prazosin saturation radioligand binding shows that mutation of leucine 132 to serine does not alter the ligand-binding properties of the α_1A-AR. Results in B show the means ± S.E. of three experiments performed in triplicate. C, the L132S mutation markedly inhibits α_1A-AR functional coupling, as measured by phenylephrine-stimulated PI hydrolysis. Results in C are normalized to the response of WT α_1A-AR to 100 μm phenylephrine (maximal response) and presented as the means ± S.E. of four experiments performed in triplicate.

FIGURE 4.

A model to suggest the involvement of IL2 in hydrophobichydrophobic interactions between GPCR and G-protein. A, docking analysis reveals that the IL2 of GPR54/β2-AR comes into close proximity to the GTPase domain of the activated Gα_q subunit. B, Pro¹³⁸ of the β2-AR positions Phe¹³⁹ so that a productive hydrophobic interaction face is formed with highly conserved Phe²²⁰, Val²²³, Trp²⁶³, and Phe²⁶⁴ residues of Gα_q. C, the third intracellular loop of GPR54/β2-AR also comes into close contact with the α4/β6 loop of the Gα_q subunit. D, mutational analysis of GPR54 demonstrates that the presence of a highly hydrophilic, acidic, or basic amino acid at position 148 inhibits functional coupling, as measured by PI hydrolysis. Data are normalized to the WT response to 1 μm KP-10, which is set as 100%. Results are expressed as the means ± S.E. of four experiments performed in triplicate.

FIGURE 5.

The L148S mutation does not abolish G-protein binding, and overexpression of G-proteins partially rescues the functional deficits of IL2-10 mutants. A, in E. coli, both WT and L148S mutant GST-hGPR54 IL2 fusion proteins co-purify with Gα_q. GST alone does not co-purify with Gα_q. B, co-immunoprecipitation experiments reveal that both TAP-WT hGPR54 and TAP-L148S hGPR54 proteins pull down Gα_q and Gα_15/16. C, overexpression of Gα_15/16 DNA in L148S hGPR54-GFP cells leads to a concentration-dependent rescue of functional coupling, as measured by PI hydrolysis. L148S hGPR54 results (C) are normalized to the WT response to 1 μm KP-10, which was set at 100%. Inset, Western blot shows that increasing the amount of Gα_15/16 DNA transfected increases the relative levels of Gα_15/16 protein in L148S hGPR54-GFP cells. D, co-overexpression of L132S α_1A-AR and Gα_q modestly rescues the deficit in PI hydrolysis. The results presented in A and B are representative of three experiments, and C and D show the means ± S.E. of three experiments performed in triplicate.

FIGURE 6.

The L148S mutation abolishes the agonist-induced catalytic activation of Gα_q by GPR54. A, schematic demonstrating that under basal conditions, FRET occurs between the Gα_q-CFP and Gβ₁-YFP proteins that are docked to GPR54. B, representative confocal microscopy images showing the membrane localization of Gα_q-CFP and Gβ₁-YFP in both WT hGPR54 (top) and L148S hGPR54 (bottom) cells. C, data from a representative WT hGPR54 cell demonstrating that stimulation with 100 nm KP-10 causes a decrease in FRET between YFP and CFP. D, averaged FRET data from 11-13 cells from three distinct experiments expressing either WT or L148S hGPR54. WT hGPR54 shows a robust decrease in FRET in response to KP-10, whereas L148S hGPR54 fails to respond. For clarity in display, data are pooled in 2.5-5 bins.