A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK).
HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7 kb 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 (Gbeta1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gbeta1-C144 protein was expressed in E. coli BL21 and Anti-Gbeta1 serum was raised with purified Gbeta1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gbeta1 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 micromol/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.
Mesh Terms:
Animals, Antigens, Surface, Blotting, Western, COS Cells, Cell Line, Cercopithecus aethiops, Cytoplasm, DNA, Complementary, Escherichia coli, Extracellular Signal-Regulated MAP Kinases, Female, GTP-Binding Protein beta Subunits, GTP-Binding Proteins, Humans, Immunoprecipitation, MAP Kinase Signaling System, Male, Microscopy, Fluorescence, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Ovary, Plasmids, Protein Kinase C, Protein Structure, Tertiary, Recombinant Proteins, Signal Transduction, Spermatogenesis, Spermatozoa, Testis, Tissue Distribution, Transfection, Two-Hybrid System Techniques
Animals, Antigens, Surface, Blotting, Western, COS Cells, Cell Line, Cercopithecus aethiops, Cytoplasm, DNA, Complementary, Escherichia coli, Extracellular Signal-Regulated MAP Kinases, Female, GTP-Binding Protein beta Subunits, GTP-Binding Proteins, Humans, Immunoprecipitation, MAP Kinase Signaling System, Male, Microscopy, Fluorescence, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Ovary, Plasmids, Protein Kinase C, Protein Structure, Tertiary, Recombinant Proteins, Signal Transduction, Spermatogenesis, Spermatozoa, Testis, Tissue Distribution, Transfection, Two-Hybrid System Techniques
Front. Biosci.
Date: Dec. 22, 2005
PubMed ID: 16368546
View in: Pubmed Google Scholar
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