Nonmuscle myosin IIA (myosin heavy polypeptide 9): a novel class of signal transducer mediating the activation of G alpha h/phospholipase C-delta 1 pathway.
The dimeric Gh protein is comprised of alpha (tissue transglutaminase) and beta (Calreticulin) subunits and known to be associated with FSH-, oxytocin-, or epinephrine-receptors/functions in their respective target cells. After establishing the FSH-induced activation of G alpha h/phospholipase C (PLC)-delta 1 pathway in rat Sertoli cells (SCs), we have attempted to identify a possible G alpha h-coupled novel FSH receptor (FSH-R). Remarkably, a protein with approximately 240-kDa molecular mass was coimmunoprecipitated with G alpha h in the fractionated membrane proteins of rat SCs. The protein was identified as myosin heavy polypeptide 9 (MyH9) by mass spectrometric analysis and immunoblotting. In addition, immunoprecipitation analysis reveals that MyH9 is constitutively associated with classical Gs-coupled FSH-R and inactive GDP-bound G alpha h at resting state of rat SCs, but did not interact with FSH directly as judged by Far-Western analysis. Upon the stimulation of higher levels of extracellular FSH (>1000 IU/liter), classical FSH-R induces the phosphorylation of MyH9, the dissociation of active GTP-bound G alpha h from FSH-R:MyH9 complexes, and the elicitation of G alpha h/PLC-delta 1 pathway-dependent Ca(2+)-influx in rat SCs. Furthermore, the specific inhibition of MyH9 ATPase activity with Blebbistatin dose-dependently suppressed FSH-induced G alpha h/PLC-delta 1 signaling and Ca(2+)-influx, but not intracellular cAMP accumulation in rat SCs, implying that MyH9 mediates FSH-induced activation of G alpha h/PLC-delta 1/IP(3)/Ca(2+)-influx pathway in rat SCs. This is the first to demonstrate that the filament protein MyH9 constitutively forms a ternary complex with FSH-R and inactive GDP-bound G alpha h. At higher FSH levels, this ternary complex executes an alternative signaling of classical Gs-coupled FSH-R through activating a Gs/cAMP-independent, G alpha h/PLC-delta 1 pathway in rat SCs.
Lin
Yuan-Feng
YF
School of Pharmacy, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Yeh
Tien-Shun
TS
Chen
Sung-Fang
SF
Tsai
Yu-Hui
YH
Chou
Chih-Ming
CM
Yang
Yi-Yuan
YY
Huang
Haw-Ming
HM
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020013:Calcium Signaling; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D054803:Phospholipase C delta / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011962:Receptors, FSH / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism*
C527821:Ghalpha protein, rat; D011962:Receptors, FSH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone; D011503:Transglutaminases; D054803:Phospholipase C delta; D019204:GTP-Binding Proteins; D024507:Nonmuscle Myosin Type IIA; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases