T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane.

Insulin-like 3 (INSL3) hormone plays a crucial role in testicular descent during embryonic development. Genetic ablation of Insl3 or its G protein-coupled receptor (GPCR) Lgr8 causes cryptorchidism in mice. Previously, we identified a nonfunctional T222P mutation of LGR8 in several human patients with testicular maldescent. Using a large population of patients and healthy controls from Italy, we have demonstrated that T222P LGR8 mutation is present only in affected patients (19 T222P/+ of 598 vs. 0/450, P < 0.0001). We have also identified a novel allele of LGR8 (R223K) found in one patient with retractile testes. Both mutations are located in the leucine-rich repeats (LRRs) of GPCR ectodomain. The expression analysis of T222P mutant receptor transfected into 293T cells revealed that the mutation severely compromised GPCR cell membrane expression. The substitution of Thr(222) with the neutral Ser or Ala, or the R223K mutation, did not alter receptor cell membrane expression or ligand-induced cAMP increase. Additional mutations, affecting first leucine in a signature LxxLxLxxN/CxL stretch of LRR (L283F), or the amino acid residues, forming the disulfide bond or coordinating calcium ion in the LDLa module (C71Y and D70Y), also rendered proteins with reduced cell surface expression. The structural alterations of both LRRs and LDLa of the ligand-binding part of LGR8 cause the inability of receptor to express on the cell surface membrane and might be responsible for the abnormal testicular phenotype in patients.