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. 2013 May 24;123(6):2421–2433. doi: 10.1172/JCI65952

Figure 7. Identification of an amino acid substitution of GPRC6A associated with decreased fertility in humans.

Figure 7

(A) T→A missense mutation in GPRC6A seen in 2 patients. (B) Localization of the F464Y substitution-mutation in GPRC6A. (C) Immunofluorescence of HEK293T cells transfected with WT (GPRC6A-MYC) or mutant (GPRC6A-MYC-F464Y) GPRC6A. Nuclei are stained with DAPI (blue), MYC-tag in green, and the membrane marker α 1 sodium potassium ATPase in red. (D) cAMP production upon osteocalcin stimulation (3 ng/ml) in HEK293T cells transfected with control (MYC-pcDNA3), WT (GPRC6A-pcDNA3), mutant (GPRC6A-mutant-pcDNA3), or both forms of GPRC6A. (E) cAMP production upon osteocalcin stimulation (3 ng/ml) in HEK293T cells transfected with WT (GPRC6A-pcDNA3), mutant (GPRC6A-mutant-pcDNA3) or both forms of GPRC6A (ratio of 1[WT]: 0.1 [mutant], 1:0.25, 1:0.5, 1:0.75, and 1:1). (F) qPCR and Western blot analysis of HEK293T cells cotransfected with WT (GPRC6A-FLAG) and mutant (GPRC6A-MYC-F464Y) GPRC6A at different ratios. (G and H) Immunofluorescence analyses of HEK293T cells cotransfected with WT and mutant GPRC6A at a ratio of (G) 1:1 (WT/mutant) and (H) 1:0.25. FLAG-tag stained in red, MYC-tag in green, and nuclei in blue (DAPI). (I) qPCR analysis of StAR expression in TM3 cells infected with pLenti6.3/V5 lentiviral vectors containing WT GPRC6A or the F464Y mutant and treated with vehicle or osteocalcin (1 ng/ml). (J) qPCR analysis of StAR, 3β-HSD, and Cyp11a expression in WT testis injected with vehicle, WT GPRC6A (LV-GPRC6A-WT), or the GPRC6A-F464Y-mutant (LV-GPRC6A-F464Y) (n = 4 for each conditions). Scale bar: 10 μm. *P < 0.05; ***P < 0.001.

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