High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs

Abstract

Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates G(q) in the absence of agonist, as assessed through guanosine 5'-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with μ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.

FIGURE 1.

Reconstitution of the purified GHS-R1a in lipid discs. The affinity-purified GHS-R1a after reconstitution in lipid discs was loaded onto a Superdex S200HR column (10 × 400 mm) and elution was carried out as described under “Experimental Procedures”; inset, blue native-PAGE followed by Coomassie Blue staining of the particles in peak 2. Lane 1, molecular weight markers, lane 2, GHS-R1a -containing discs (peak 2 pooled fractions). The molecular mass of the markers is indicated in Da.

FIGURE 2.

Purified GHS-R1a binds its ligands. A, FRET-monitored binding of an FITC-labeled ghrelin peptide (JMV 4946) to Alexa Fluor 350-labeled GHS-R1a assembled in lipid discs. The binding data are presented as variations in FRET ratio as a function of JMV 4946 concentration. Inset, fluorescence emission spectrum obtained for Alexa Fluor 350-labeled GHS-R1a in lipid discs in the presence of saturating concentrations in JMV 4946 (solid line) and in the presence of JMV 4946 and an excess of the JMV3002 antagonist (dotted line). B, FRET-monitored competition experiments. JMV 4946 was displaced from its binding site in the presence of increasing concentrations in the JMV3002 antagonist (open circles), the JMV3018 antagonist (closed circles), or the SPA inverse agonist (open squares). Data are presented as the mean ± S.D. from three independent experiments.

FIGURE 3.

Isolated GHS-R1a triggers G_q protein activation. A, IP1 production induced by the GHS-R1a receptor expressed in HEK293T cells in the absence of ligand, in the presence of the inverse agonist SPA, or in the presence of the full agonist ghrelin. IP1 accumulation is expressed as the percentage of the maximal ghrelin response and 0 represents the IP1 accumulation in cells transfected with an empty vector. The figure is representative of one experiment of three. B, BODIPY FL GTPγS binding to Gα_q protein induced by GHS-R1a in the absence of ligand, in the presence of SPA, or in the presence of ghrelin. Data are presented as the percentage of maximum BODIPY FL fluorescence change measured in the presence of ghrelin and represent the mean ± S.D. from three independent experiments.

FIGURE 4.

Purified GHS-R1a recruits arrestin in an agonist-dependent manner. A, ligand-dependent arrestin recruitment by GHS-R1a in HEK293T cells. BRET signal (ratio 530 nm/485 nm) is expressed as a percentage of the maximal ghrelin response and 0 represents BRET signal of mock cells transfected with an empty vector. The figure is one experiment representative of three. B, changes in emission intensity of bimane-labeled arrestin induced by GHS-R1a in the absence of ligand, in the presence of ghrelin, or in the presence of SPA, ghrelin, Gαβγ_q, and Gαβγ_q and GTPγS. Data are presented as the percentage of maximum bimane fluorescence change measured in the presence of ghrelin and represent the mean ± S.D. from three independent experiments.

FIGURE 5.

Isolated GHS-R1a binds μ-AP2. A, changes in emission intensity of bimane-labeled μ-AP2 induced by GHS-R1a in the absence of ligand, with 1 μm SPA, or 1 μm ghrelin. Data are presented as the percentage of maximum bimane fluorescence change measured in the presence of ghrelin and represent the mean ± S.D. from three independent experiments. B, changes in emission intensity of bimane-labeled μ-AP2 induced by GHS-R1a in the presence of purified arrestin-2 and in the absence of ligand, in the presence of SPA, or in the presence ghrelin. Data are presented as the percentage of maximum bimane fluorescence change measured in the presence of ghrelin and in the absence of arrestin-2 and represent the mean ± S.D. from three independent experiments.

FIGURE 6.

Ligand binding affects GHS-R1a conformation. Intrinsic fluorescence spectra of GHS-R1a in lipid discs in the absence and presence of ligands. Empty discs or monomeric GHS-R1a in lipid discs were incubated with or without the drug to be tested (MK0677, SPA; 1 μm). Data are presented as the intensity of tryptophan emission at its maximum normalized to that in the absence of the ligand. Inset, fluorescence emission spectrum of the ligand-free receptor in lipid discs. Data are presented as the mean ± S.D. from three independent experiments.

FIGURE 7.

Schematic representation of the functional behavior of the purified GHS-R1a. A, in the absence of ligand, the purified receptor activates Gα_q and recruits μ-AP2, whereas no significant basal recruitment of arrestin occurs. B, in the presence of the ghrelin agonist, Gα_q is further activated, arrestin-2 is recruited, and μ-AP2 still interacts with the receptor; in contrast, binding of the inverse agonist SPA significantly reduces the receptor constitutive activity and dissociates the complex with μ-AP2. The width of the arrow in the receptor·G_q complexes represents the extent in G protein activation.