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^-11M for T release (respectively P = 0,041 10^-13M; P = 0,032 10^-12M; P = 0,008 10^-11M vs basal) and for 10^-12 to 10^-10M for insulin (respectively P = 0,041 10^-12M; P = 0,007 10^-11M; P = 0,047 10^-10M; 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.