Rescue of expression and signaling of human luteinizing hormone G protein-coupled receptor mutants with an allosterically binding small-molecule agonist

Abstract

Naturally occurring mutations of G protein-coupled receptors (GPCRs) causing misfolding and failure to traffic to the cell surface can result in disease states. Some small-molecule orthosteric ligands can rescue such misfolded receptors, presumably by facilitating their correct folding and shuttling to the plasma membrane. Here we show that a cell-permeant, allosterically binding small-molecule agonist (Org 42599) rescues the folding and cell surface expression, and therefore target cell signaling, of mutant human luteinizing hormone (LH) receptors (A593P and S616Y) that cause Leydig cell hypoplasia in man. Both mutant receptors were retained in the cytoplasm whereas WT receptor localized at the cell membrane, and binding of LH to cells expressing the mutant receptors was markedly lower than to those expressing the WT receptor. Incubation with Org 42599 increased mutant receptor expression, cell surface localization, and the proportion of mutant receptor in the mature glycosylated form. Importantly, although LH stimulated little (S616Y) or no (A593P) activation of cells expressing mutant receptors, incubation of cells with Org 42599 facilitated rescue of expression and stimulation by the native ligand, LH. Although Org 42599 could activate these receptors, it could not displace (125)I-labeled human LH binding to the WT receptor, indicating that it acts in an allosteric manner. Here we demonstrate a small-molecule GPCR allosteric agonist that functionally rescues intracellularly retained mutant LH receptors by facilitating their cell surface expression. This approach may have application for treatment of infertile patients bearing such mutations and, more broadly, for other misfolded GPCR mutants resulting in human pathologic processes.

Fig. 1.

Cellular localizations of mutant LH receptors are altered after incubation with Org 42599. Cells expressing WT, A593P mutant, or S616Y mutant LH receptors were incubated in the presence of vehicle (Left) or 1 μM Org 42599 (Right) for 24 h before fixation, fluorescent labeling, and confocal imaging (Materials and Methods). LH receptors (myc tagged) are labeled in green and cell nuclei marker (DAPI) in blue. Images are from one experiment and are representative of three independent experiments with similar results. (Scale bar: 10 μm.)

Fig. 2.

LH elicits little or no activation of cells expressing mutant LH receptors. Measurement of cAMP accumulation by cAMP ELISA after 1 h stimulation (A) or CRE-luciferase reporter gene activation after 24 h stimulation (B) in cells expressing WT (●), A593P mutant (▪), or S616Y (▲) mutant LH receptors over a range of concentrations of LH (Materials and Methods). Data were fitted by sigmoidal dose–response curves with Hill coefficients of unity. Data are presented as fold versus basal values for each receptor and are mean ± SEM from at least three independent experiments.

Fig. 3.

Org 42599 elicits robust activation of cells expressing WT and mutant LH receptors. Measurement of cAMP accumulation by CRE-luciferase reporter gene activation after 24 h stimulation in cells expressing WT (●), A593P mutant (▪), or S616Y (▲) mutant LH receptors was determined over a range of concentrations of Org 42599 (Materials and Methods). Data were fitted by sigmoidal dose–response curves with Hill coefficients of unity. Data are presented as fold versus basal values for each receptor and are mean ± SEM from at least three independent experiments.

Fig. 4.

Binding of ¹²⁵I-hLH to cell-surface mutant LH receptors is increased in a time- and concentration- dependent manner after incubation with Org 42599. Binding of ¹²⁵I-hLH to cells expressing WT, A593P mutant, or S616Y mutant LH receptors was measured after incubation with Org 42599 (1 μM) for a range of incubation times (A) or for 24 h with a range of concentrations of Org 42599 (B). After incubation with Org 42599, cells were washed once for 1 h with Complete media before incubation with radioligand. Data are presented as fold versus binding in the absence of Org 42599 treatment and are mean ± SEM from at least three independent experiments. Note WT receptor binding is not increased by Org 42599 treatment (1.0-fold vs. vehicle control), whereas mutant receptors show a time- and concentration- dependent increase in binding after incubation with Org 42599. *P < 0.05 and **P < 0.01 (one-sample t test) for comparison with no change compared with vehicle control (1.0-fold change, dotted line).

Fig. 5.

LH stimulation of cells expressing S616Y mutant LH receptors is increased by preincubation with Org 42599. cAMP accumulation was measured by cAMP ELISA after stimulation of cells expressing S616Y mutant LH receptors with LH (3 nM) for 1 h at 37 °C after preincubation in the presence or absence of Org 42599 (0.1 μM) for 2 to 6 h and washing once for 1 h (SI Materials and Methods). Data are mean ± SEM from three independent experiments and are presented as percentage of the maximum LH response obtained in the absence of Org 42599 incubation.