Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone.
The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPgammaS revealed a class of binding sites with an apparent Kd value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Galphai-3, Galpha0, Galphas, and Galphaq were not increased after aldosterone. The patterns of small Mr G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [alpha-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of Mr 59.5 kDa.
Sariban-Sohraby
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S
Laboratoire de Physiologie, Université Libre de Bruxelles, 1070 Brussels, Belgium.
Svoboda
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M
Mies
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F
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D037342:Pertussis Toxin; D019920:Photoaffinity Labels; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014982:Xenopus laevis
D008565:Membrane Proteins; D019920:Photoaffinity Labels; D012333:RNA, Messenger; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000450:Aldosterone; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D012260:Ribonucleases; D019204:GTP-Binding Proteins