Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated by RF-amide-related peptides through the Galpha q/11 pathway

Abstract

MrgA1 and MrgC11 belong to a recently identified family of orphan G-protein coupled receptors, called mrgs (mas-related genes). They are only expressed in a specific subset of sensory neurons that are known to detect painful stimuli. However, the precise physiological function of Mrg receptors and their underlying mechanisms of signal transduction are not known. We therefore have screened a series of neuropeptides against human embryonic kidney (HEK) 293 cells that stably express either MrgA1 or MrgC11 to identify ligands and/or agonists. MrgA1- or MrgC11-specific agonists stimulated dose-dependent increases in intracellular free Ca(2+) in a pertussis toxin-insensitive manner, but failed to alter basal or forskolin-stimulated levels of intracellular cAMP. Furthermore, studies using embryonic fibroblasts derived from various Galpha protein knockout mice demonstrated that both the MrgA1 and MrgC11 receptors are coupled to the Galpha(q/11) signaling pathway. Screening of neuropeptides identified surrogate agonists, most of these peptides included a common C-terminal -RF(Y)G or -RF(Y) amide motif. Structure-function studies suggest that endogenous ligands of Mrg receptors are likely to be RF(Y)G and/or RF(Y) amide-related peptides and that postprocessing of these peptides may serve to determine Mrg receptor-ligand specificity. The differences in ligand specificity also suggest functional diversity amongst the Mrg receptors.

Fig 1.

Sequence comparisons of mouse MrgA1, MrgC11, and human MrgX1 and expression of MrgC11. (A) Alignment of the amino acid sequence of mouse MrgA1 (mMrgA1), mouse MrgC11 (mMrgC11), and human MrgX1 (hMrgX1). Residues shaded in black are identical in >50% of the proteins and residues shaded in gray indicate conservative substitutions. The 7 transmembrane domains (TM1–7) are over-lined. (B and C) In situ hybridization with cRNA riboprobes detecting mMrgC11 in newborn (B) and adult (C) DRG neurons. (D) Double label in situ with mMrgC11 probe (red) and staining with fluorescent lectin IB4 (green) in adult mouse DRG neuron.

Fig 2.

Calcium signaling in HEK-MrgA1 (A–C) and HEK-MrgC11 (D–F). Cells loaded with Fura-2/AM were stimulated with each agonist, and fluorescence was recorded. Graphs represent an average plot of [Ca²⁺]_i measurements versus time (in s) in a minimum of 8 cells from representative experiments. Individual data points represent images taken at 0.8-s intervals. (A and D) (○), the active phospholipase C inhibitor blocked agonist-induced rise in [Ca²⁺]_i. However, (•), the inactive analogue, did not affect FLRFa or γ2-MSH-induced Ca²⁺ mobilization. After a 10-min pretreatment with and , each agonist was added. (B and E) Extracellular [Ca²⁺] dependency of Ca²⁺ mobilization. Cells were preincubated for 2 min with 2 mM EGTA (○) or normal medium containing 1.2 mM calcium (•), and then 3 μM FLRFa or 1 μM γ2-MSH were added. (C and F) TG prevents the agonist-evoked increase of [Ca²⁺]_i in HEK-MrgA1 (C) and HEK-MrgC11 (F). In the presence of 2 mM EGTA, TG (1 μM final concentration) was added to deplete internal Ca²⁺ store.

Fig 3.

Internalization of MrgA1-GFP (A and B) and MrgC11-GFP (C and D) was induced by 3 μM FLRFa and 1 μM γ2-MSH, respectively. (A and C) Serum-starved (>4 h) HEK-MrgA1 or HEK-MrgC11 cells. (B and D) HEK-MrgA1 or HEK-MrgC11 were treated with the indicated agonists for 30 min at 37°C. Results are representative of three independent experiments, and the arrow indicates the internalization process.

Fig 4.

Heterotrimeric G protein coupling of MrgA1 and MrgC11. (A and D) FLRFa or γ2-MSH dose-dependently stimulates intracellular calcium mobilization in HEK-MrgA1 or HEK-MrgC11 in the absence (•) or presence (▪) of PTX (16 h, 100 ng/ml). All results shown are the mean of triplicate determination ± SEM. (B and E) Effect of Gα subunit KO on [Ca²⁺]_i mobilization. KO MEFs were derived from KO mice at embryonic 8.5 and 9.5 days. Gα_12/13 KO MEF (▴) and Gα_q/11 KO MEF (○) were transfected with the cDNAs encoding the MrgA1-GFP (B) or the MrgC11-GFP (E). FLRFa or γ2-MSH evoked [Ca²⁺]_i responses were completely ablogated in Gα_q/11 KO MEF expressing MrgA1-GFP (B) or MrgC11-GFP (E). However, cotransfection (▵) of wild-type Gα_q plus MrgA1-GFP or MrgC11-GFP in Gα_q/11 double KO MEF restored responsiveness to FLRFa or γ2-MSH, respectively. Positively transfected cells were selected by their green fluorescence excited at 480 nm (GFP-positive cells). On the same field, cells that did not express GFP (GFP-negative cells) were selected as internal control. (C and F) cAMP production in HEK-MrgA1 (C) or HEK-MrgC11 (F). Cells were stimulated with various concentrations of FLRFa or γ2-MSH in the presence or absence of 10 μM forskolin as described in Materials and Methods. Each value represents the mean ± SEM for three independent experiments.