Activation of the calcium-permeable cation channel CD20 by alpha subunits of the Gi protein.
When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducted to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 channel by IGF-I required GTP, Mg2+, and ATP in the bath solution, and removal of either GTP or ATP attenuated the activation. Non-hydrolyzable ATP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked the activation of the channel by IGF-I. The CD20 channel was also activated by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required for the activation. Addition of a preparation of Gi/Go holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP-bound form of recombinant Gi2 alpha subunit purified from Sf9 cells. The Gi3 alpha subunit was less effective, and the Gi1 alpha subunit had no effect. Purified recombinant beta1gamma2 subunits did not affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against Gi2 alpha subunit. These findings indicate that the CD20 channel expressed in Balb/c 3T3 cells is activated by the IGF-I receptor via the alpha subunits of heterotrimeric G proteins.
Kanzaki
M
M
Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371, Japan.
Lindorfer
M A
MA
Garrison
J C
JC
Kojima
I
I
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D018951:Antigens, CD20 / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000378:metabolism; D002417:Cattle; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018951:Antigens, CD20; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007334:Insulin-Like Growth Factor I; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins