[Frontiers in Bioscience 11, 1679-1689, May 1, 2006]
A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK)
Ning Liu 1, Yuan Qiao 1, Congli Cai 1, Wen Lin 1, Jianchao Zhang 2, Shiying Miao 1, Shudong Zong 2, S.S.Koide 3 and Linfang Wang 1
1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, 100005, People's Republic of China,2 National Research Institute for Family Planning, WHO Collaboration Center for Research in Human Reproduction, 12 Da Hui Si, Beijing 100081, People's Republic of China, 3Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021, USA
TABLE OF CONTENTS
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 l 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 Gb 1 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 Ámol/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.