1. Academic Validation
  2. SHBG141-161 Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and β-Langerhans cell lines

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

  • Sci Rep. 2019 Dec 19;9(1):19432. doi: 10.1038/s41598-019-55941-x.
Luca De Toni 1 Diego Guidolin 2 Vincenzo De Filippis 3 Daniele Peterle 3 Maria Santa Rocca 1 4 Andrea Di Nisio 1 Maurizio De Rocco Ponce 1 Carlo Foresta 5
Affiliations

Affiliations

  • 1 University of Padova, Department of Medicine and Unit of Andrology and Reproductive Medicine, 35128, Padova, Italy.
  • 2 Unversity of Padova, Department of Neuroscience and Section of Anatomy, 35128, Padova, Italy.
  • 3 University of Padova, Department of Pharmaceutical and Pharmacological Sciences, 35131, Padova, Italy.
  • 4 Familial Cancer Clinic, Veneto Institute of Oncology (IOV-IRCCS), 35128, Padova, Italy.
  • 5 University of Padova, Department of Medicine and Unit of Andrology and Reproductive Medicine, 35128, Padova, Italy. carlo.foresta@unipd.it.
Abstract

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 (SHBG141-161) on GPRC6A. The binding of SHBG141-161 to GPRC6A and downstream dissociation from Gαi(GDP) protein was computationally modelled. SHBG141-161 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 SHBG141-161 was assessed on Leydig MA-10 and Langerhans β-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and Insulin. SHBG141-161 was predicted to bind to GPRC6A and to reduce the affinity for Gαi(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG141-161 were confirmed by CD and displacement experiments. SHBG141-161 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 SHBG141-161, suggesting agonist specificity. SHBG141-161 showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorders.

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