SHBG141–161 Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and β-Langerhans cell lines

GPRC6A is acknowledged as a major regulator of energy metabolism and male fertility through the action of undercarboxylated osteocalcin (ucOCN), representing a possible therapeutic target. We recently showed that the sex hormone-binding globulin (SHBG) binds to GPRC6A through the likely involvement of the 141–161 domain. To confirm this model, here we investigated the possible binding and agonist activity of SHBG(141–161) domain-peptide (SHBG(141–161)) on GPRC6A. The binding of SHBG(141–161) to GPRC6A and downstream dissociation from G(αi)(GDP) protein was computationally modelled. SHBG(141–161) was obtained by solid-phase synthesis, characterized by circular dichroism (CD) and the receptor binding was assessed by displacement of ucOCN on HEK-293 cells transfected with GPRC6A gene. Agonist activity of SHBG(141–161) was assessed on Leydig MA-10 and Langerhans β-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and insulin. SHBG(141–161) was predicted to bind to GPRC6A and to reduce the affinity for G(αi)(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG(141–161) were confirmed by CD and displacement experiments. SHBG(141–161) stimulated cell secretion of T and insulin, with dose dependency from 10(−13) to 10(−11)M for T release (respectively P = 0,041 10(−13)M; P = 0,032 10(−12)M; P = 0,008 10(−11)M vs basal) and for 10(−12) to 10(−10)M for insulin (respectively P = 0,041 10(−12)M; P = 0,007 10(−11)M; P = 0,047 10(−10)M; P = 0,045 vs basal). Blockade with anti GPRC6A IgG abolished the response to SHBG(141-161), suggesting agonist specificity. SHBG(141–161) showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorders.