G-protein mRNA levels during adipocyte differentiation.
G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.
Watkins
D C
DC
Department of Pharmacology, State University of New York, Stony Brook 11794-8651.
Rapiejko
P J
PJ
Ros
M
M
Wang
H Y
HY
Malbon
C C
CC
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D015151:Immunoblotting; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D037342:Pertussis Toxin; D012333:RNA, Messenger / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D012333:RNA, Messenger; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D003907:Dexamethasone; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015056:1-Methyl-3-isobutylxanthine