Pharmacological characterization of mouse GPRC6A, an L-alpha-amino-acid receptor modulated by divalent cations

Abstract

Background and purpose: GPRC6A is a novel member of family C of G protein-coupled receptors with so far unknown function. We have recently described both human and mouse GPRC6A as receptors for L-alpha-amino acids. To date, functional characterization of wild-type GPRC6A has been impaired by the lack of activity in quantitative functional assays. The aim of this study was thus to develop such an assay and extend the pharmacological characterization of GPRC6A.

Experimental approach: We have engineered a novel cell-based inositol phosphate turnover assay for wild-type mouse GPRC6A based on transient co-expression with the promiscuous Galpha(qG66D) protein, known to increase receptor signalling sensitivity. This assay allowed for measurements of L-alpha-amino acid potencies. Furthermore, in combination with an assay measuring inward currents at Ca(2+)-activated chloride channels in Xenopus oocytes, the divalent cation-sensing ability of the receptor was examined.

Key results: Using our novel assay, we demonstrate that the basic L-alpha-amino acids ornithine, lysine, and arginine are the most potent agonists at wild-type mouse GPRC6A. Using two different assay systems, we show that divalent cations do not activate the G(q) signalling pathway of mouse GPRC6A per se but positively modulate the amino-acid response.

Conclusions and implications: This is the first reported assay for a wild-type GPRC6A successfully applied for quantitative pharmacological characterization of amino acid and divalent cation responses at mouse GPRC6A. The assay enables further search for GPRC6A ligands such as allosteric modulators, which may provide essential information about the physiological function of GPRC6A.

Figure 1

Ability of mGPRC6A to signal through various Gα proteins. tsA cells co-transfected with either mGPRC6A or empty vector, and the indicated Gα proteins were stimulated with 1 mM L-Orn for 30 min at 37°C. The formation of IP was determined as described under Methods. Results are shown as fold increase in IP production upon ligand stimulation, normalized to non-stimulated cells. Data are means±s.d. of triplicate determinations of a single representative experiment. Two additional experiments gave similar results. An unpaired, one-tailed t-test was performed to determine whether each observed response was significantly larger than the corresponding control: ^***P<0.001.

Figure 2

Ability of mGPRC6A to (a) activate or (b) inhibit cAMP formation. tsA cells transfected with mGPRC6A, GLP-1, GABA_B(1b,2) or empty vector were stimulated with the indicated ligands for 10 min at 37°C in either the absence (a) or presence (b) of 10 μM forskolin. The formation of cAMP was determined as described under Methods. Results are shown (a) as fold increase in cAMP production upon ligand stimulation, normalized to non-stimulated cells and (b) as percent cAMP concentration compared with non-stimulated cells. Data are means±s.d. of triplicate determinations of a single representative experiment. Two additional experiments gave similar results. Asterisks indicate significant stimulation (a) or inhibition (b) compared with the control: ^**P<0.01 (ANOVA followed by Dunnett's test).

Figure 3

Representative traces obtained from two-electrode voltage clamp recordings on Xenopus oocytes that are either uninjected or injected with cRNA encoding mGPRC6A. (a) No responses could be detected at Ca²⁺-activated chloride channels upon application of 50 mM CaCl₂ alone in either mGPRC6A-expressing (N=9) or uninjected Xenopus oocytes (N=6). The divalent cation-free Ringer's solution was supplemented with 1.8 mM MgCl₂. (b) No responses could be detected at Ca²⁺-activated chloride channels upon application of 50 mM MgCl₂ alone in either mGPRC6A-expressing (N=5) or uninjected Xenopus oocytes (N=4). The divalent cation-free Ringer's solution was supplemented with 1.8 mM CaCl₂. (c) L-Orn (100 μM) did not activate mGPRC6A in the absence of divalent cations. Co-application of 100 μM L-Orn and either 1.8 mM CaCl₂ (N=7) or 1.8 mM MgCl₂ (N=8) restored mGPRC6A-mediated responses. Recordings were first made in divalent cation-free Ringer's solution, and then the oocytes were allowed to equilibrate for 5 min in divalent cation-free Ringer's solution supplemented with either 1.8 mM CaCl₂ or 1.8 mM MgCl₂. The recordings shown were from two different oocytes. The part of the traces recorded in divalent cation-free Ringer's solution without CaCl₂ or MgCl₂ was corrected for drift of baseline current, as described in the Methods section. No mGPRC6A-mediated responses were observed upon application of either 1.8 mM CaCl₂ alone or 1.8 mM MgCl₂ alone to mGPRC6A-expressing oocytes voltage clamped in divalent cation-free Ringer's solution. (d) In both mGPRC6A-expressing (N=3) and uninjected (N=3) oocytes, 10 μM LPA was able to evoke responses at Ca²⁺-activated chloride channels mediated by endogenously expressed GPCRs in the absence of divalent cations. The recordings shown were from two different oocytes. The traces were recorded in divalent cation-free Ringer's solution and corrected for drift of baseline current as described in the Methods section. (e) Recordings were made in a total of 12 mGPRC6A-expressing oocytes. Of these, six oocytes did not respond to application of 1 mM L-Orn in divalent cation-free Ringer's solution supplemented with 1 mM EDTA (N=6). However, six oocytes showed small responses to 1 mM L-Orn under the same conditions (N=6). Recordings were first made in divalent cation-free Ringer's solution supplemented with 1 mM EDTA, and the oocytes were then allowed to equilibrate for 5 min in divalent cation-free Ringer's solution supplemented with 1 mM EDTA and 2.8 mM CaCl₂. The recordings shown were from two different oocytes. The part of the traces recorded in divalent cation-free Ringer's solution without CaCl₂ was corrected for drift of baseline current, as described in the Methods section.

Figure 4

mGPRC6A is not activated by Mg²⁺ per se. tsA cells co-transfected with mGPRC6A and Gα_qG66D or rat CaR were incubated with either 40 mM Mg²⁺ or 10 mM L-Orn for 30 min at 37°C. Mock-transfected cells and cells co-transfected with empty vector and Gα_qG66D were used as control. All experiments were performed in the presence of 1 mM Ca²⁺. The formation of IP was determined as described under Methods. Results are shown as fold increase in IP production, normalized to the control. Data are means±s.d. of triplicate determinations of a single representative experiment. Two additional experiments gave similar results. Asterisks indicate significant differences compared with the control: ^**P<0.01 (ANOVA followed by Dunnett's test).

Figure 5

Measurement of IP production as a function of extracellular Mg²⁺ concentration at mGPRC6A and 5-HT_2C. (a) Response to Mg²⁺ measured in cells transiently co-expressing mGPRC6A and Gα_qG66D in the absence or presence of 30 μM L-Orn (∼EC₂₅). The response to maximal concentration (10 mM) of L-Orn in the presence of 1 mM Mg²⁺ was used as control. (b) Response to Mg²⁺ measured in cells transiently expressing the 5-HT_2C receptor, in the absence or presence of 2 nM 5-HT (∼EC₂₅). The response to maximal concentration (1 μM) of 5-HT in the presence of 1 mM Mg²⁺ was used as control. All experiments were performed in the presence of 1 mM Ca²⁺. The formation of IP was determined as described under Methods. Results are expressed as CPM and are means±s.d. of triplicate determinations of a single representative experiment. Two additional experiments gave similar results. Asterisks indicate significant differences from the measurements where only Ca²⁺ was present: ^**P<0.01 (ANOVA followed by Dunnett's test).

Figure 6

Concentration–response curves of ligands at mGPRC6A and 5-HT_2C, respectively, in the presence of varying concentrations of extracellular Mg²⁺. (a) Concentration–response curves of L-Orn with 0, 1, 5, 20, and 40 mM Mg²⁺ present generated from stimulation of IP formation in tsA cells transiently co-expressing mGPRC6A and Gα_qG66D. (b) Concentration–response curves of 5-HT with 0, 1, and 40 mM Mg²⁺ present generated from stimulation of IP formation in tsA cells transiently expressing the 5-HT_2C receptor. All experiments were performed in the presence of 1 mM Ca²⁺. The formation of IP was determined as described under Methods. Results are expressed as CPM and are means±s.d. of triplicate determinations of a single representative experiment. Two additional experiments gave similar results.