Osteocalcin and Sex Hormone Binding Globulin Compete on a Specific Binding Site of GPRC6A.
The undercarboxylated form of osteocalcin (ucOC) regulates male fertility and energy metabolism, acting through the G protein-coupled receptor (GPRC)6A, thus forming a new pancreas-bone-testis axis. Recently, GPRC6A has also been suggested to mediate the nongenomic responses of free testosterone (T). However, these data did not consider the physiological scenario, where circulating T is mainly bound to sex hormone-binding globulin (SHBG) and only a small percentage circulates freely in the blood. Here, by the use of computational modelling, we document the existence of similar structural moieties between ucOC and SHBG that are predicted to bind to GPRC6A at docking analysis. This hypothesis of competition was assessed by binding experiments on human embryonic kidney-293 cells transfected with human GPRC6A gene. Unliganded SHBG specifically bound the membrane of human embryonic kidney-293 cells transfected with GPRC6A and was displaced by ucOC when coincubated at 100-fold molar excess. Furthermore, specific downstream Erk1/2 phosphorylation after stimulation of GPRC6A with ucOC was significantly blunted by 100-fold molar excess of unliganded SHBG. Intriguingly previous incubation with unliganded SHBG, followed by incubation with T, induced Erk1/2 phosphorylation in a dose-dependent manner. Neither binding nor stimulating activities were shown for SHBG saturated with T. Experiments on mutation constructs of GPRC6A strengthened the hypothesis of a common binding site of ucOC and SHBG. Given the role of GPRC6A on energy metabolism, these data agree with epidemiological association between SHBG levels and insulin sensitivity, suggest GPRC6A as a likely SHBG receptor, and add bases for the possible regulation of androgen activity in a nonsteroidal manner.
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Luca
L
De Toni
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Diego
D
Guidolin
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Vincenzo
V
De Filippis
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Simone
S
Tescari
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Giacomo
G
Strapazzon
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Maria
M
Santa Rocca
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Alberto
A
Ferlin
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Mario
M
Plebani
Department of Medicine (L.D.T., M.S.R., A.F., C.F.), Unit of Andrology and Reproductive Medicine, University of Padova and Department of Laboratory Medicine (M.P.), University-Hospital, 35128 Padova, Italy; Department of Molecular Medicine (D.G.), University of Padova Medical School, 35121 Padova, Italy; Laboratory of Protein Chemistry (V.D.F., S.T.), Department of Pharmaceutical and Pharmacological Sciences, School of Medicine, University of Padova, 35131 Padova, Italy; and European Academy of Bozen/Bolzano (G.S.), Institute of Mountain Emergency Medicine, 39100 Bolzano, Italy.
Carlo
C
Foresta
D001665:Binding Sites; D004734:Energy Metabolism / Q000235:genetics / Q000502:physiology; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D015675:Osteocalcin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C488584:GPRC6A protein, human; D043562:Receptors, G-Protein-Coupled; D012738:Sex Hormone-Binding Globulin; D015675:Osteocalcin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3