A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK)

Abstract

HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7kb fragment (HSD-0.7) containing three immunological epitopes of HSD-3.8 cDNA was prepared and expressed in E. coli. Immunization of female rats with the recombinant HSD-0.7 proteins induced infertility. A cDNA fragment encoding the C-terminal 144 amino acids of human G-protein beta 1 subunit (Gβ1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gβ1-C144 protein was expressed in E.coli BL21 and Anti-Gβ1 serum was raised with purified Gβ1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gβ1 plasmids were constructed and co-transfected into human embryonal kidney 293 cells. Two proteins were localized at superimposable sites in the cytoplasm, and they formed a complex when 500 μmol/L GDP existed. Overexpression of HSD-0.7 activated the G-protein-mediated extracellular signal-regulated kinases (ERK1/2); however, the truncated fragments of HSD-0.7, which lacked either TPR domain or P-loop, lost the ability to activate the ERK1/2 pathway. Further study revealed that the activation of ERK1/2 was protein kinase C (PKC) rather than Ras dependent. These results provide evidence that HSD-3.8 present in spermatocytes and sperm may participate in spermatogenesis and fertilization process by activating the PKC-dependent ERK1/2 signal transduction pathway.