Fig 1.
Calcium mobilisation and inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells.
(A) Inositol phosphate accumulation responses to L-ornithine in FlpIn-TREx-HEK293-mGPRC6A cells with co-expression of Gαq or GαqG66D. (B) The L-ornithine response is specific to FlpIn-TREx-HEK293 cells (co-expressing GαqG66D) induced to express mGPRC6A with tetracycline, but absent in non-tetracycline induced or parental HEK293 cells. (C) GPRC6A-mediated inositol phosphate accumulation can be inhibited with the antagonist, NPS-2143 (30 μM). (D) Ca2+ mobilisation induced by various L-amino acids in FlpIn-TREx-HEK293-mGPRC6A cells co-expressing GαqG66D. The rank order of potency is L-ornithine > L-arginine = L-lysine (values quoted represent pEC50 ± SEM). Similar responses were observed with Gαq co-expression (data not shown).
Table 1.
Osteocalcin (OCN) variants used in this study.
Fig 2.
Effect of OCN on inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells.
(A) Variants of the putative GPRC6A peptide ligand osteocalcin (OCN) fail to induce inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells co-expressing GαqG66D. (B) OCN variants (40 ng/ml) do not modulate L-ornithine-stimulated inositol phosphate accumulation at mGPRC6A (OCN variants as described in Table 1).
Fig 3.
Modulation of cAMP levels and ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells.
(A) OCN variants do not stimulate cAMP accumulation in FlpIn-TREx-HEK293 cells stably expressing mGPRC6A (open circles). Neither L-ornithine nor OCN variants inhibit forksolin (3 μM)-stimulated cAMP accumulation in the same cell line (filled circles; OCN variants as described in Table 1). (B) Neither L-ornithine (1 mM) nor OCN variants (40 ng/ml) stimulate ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells; (C) a similar lack of activity was observed in cells co-expressing GαqG66D.
Fig 4.
Measurement of cellular impedance in FlpIn-TREx-HEK293-mGPRC6A cells.
(A) ATP (acting via endogenous P2Y receptors) increases cellular impedance in both tetracycline-induced and un-induced FlpIn-TREx-HEK293-mGPRC6A cells, whereas (B) L-ornithine increases cellular impedance only in tetracycline-induced cells. (C) Bovine OCN did not change cell impedance in either induced or un-induced FlpIn-TREx-HEK293-mGPRC6A cells. A similar lack of effect was shown for other OCN variants (data not shown).
Fig 5.
Determination of Gprc6a endogenous expression.
Gprc6a mRNA expression in mouse primary islets, murine β-TC6, GLUTag, MIN6 and rat INS-1(832) cells as determined by qPCR or Taqman; N.D. = not detected.
Fig 6.
GPRC6A signalling in endogenously expressing cells.
(A) L-ornithine (20 mM) significantly increases GLP-1 release from GLUTag cells (**P < 0.01 vs Vehicle, one-way ANOVA followed by Sidak’s multiple comparisons test). This effect is significantly reversed by NPS-2143 (^P < 0.05 vs. L-ornithine alone). Human synthetic OCN (0.01–10 ng/ml) did not significantly modulate GLP-1 release with or without cell washing. (B) High [glucose] significantly enhanced insulin secretion by MIN6 cells (P < 0.0001, two-way ANOVA). L-ornithine (20 mM), but not mouse synthetic OCN (acid form; 0.03–100 ng/mL), significantly increased glucose-sensitive insulin secretion (**P < 0.01 vs. Vehicle, one-way ANOVA for high [glucose] group followed by Sidak’s multiple comparison test. ^^ P < 0.01 vs. L-ornithine alone). Open bars, no glucose; filled bars, 16.7mM glucose. Data normalised to Insulin release in the presence of 16.7mM glucose.
Fig 7.
Measurement of cellular impedance in Gprc6a-negative rat INS-1(832) cells.
Concentration-dependent increases in cell index (a measure of impedance) in INS-1(832) cells (that do not express Gprc6a) with synthetic human, purified bovine and synthetic mouse OCN. Statistical analysis performed by two-way ANOVA ([OCN] and treatment); there was a significant effect of both [OCN] and treatment for all studies; *P < 0.05, **P < 0.01 vs. Vehicle by Sidak’s multiple comparison test.