Down-regulation of Gi sub-types by prolonged incubation of adipocytes with an A1 adenosine receptor agonist.
We have reported previously that prolonged incubation of adipocytes with (-)-N6-phenylisopropyl adenosine (PIA) (an A1 adenosine receptor agonist) down-regulates A1 adenosine receptors. There was a concomitant decrease in pertussis toxin catalyzed ADP-ribosylation of a 41-kDa peptide thought to be the alpha-subunit of Gi. To determine whether this represents true down-regulation of the G-protein, and if so which of the three known forms of Gi are down-regulated, we have used antipeptide antisera specific for Gi alpha-subunits. Serum SG1 recognizes alpha i1 and -2, I1C recognizes only alpha i1, and I3B recognizes alpha i3. Rat adipocytes were maintained in primary culture for up to 7 days with 0-1000 nM PIA. Crude membrane preparations were analyzed by Western blots. There was almost complete loss of alpha i1 and -3, and about 50% loss of alpha i2 from PIA-treated cells. The loss of each alpha i was detectable after 24 h with 300 nM PIA and maximal by 4 days. After 4 days, down-regulation was detectable with 3 nM and maximal with 100 nM PIA. Antiserum BN2 demonstrated approximately 50% loss of G-protein beta-subunits in cells treated with 300 nM PIA for 4 days. When cells were incubated for 4 days with 300 nM PIA and then washed to remove PIA, alpha i1, -2, and -3 and beta-subunits returned to control levels within 5 days. Antiserum CS1 detected normal amounts of both the 43- and 47-kDa forms of Gs alpha in PIA-treated cells. We conclude that Gi alpha-subunits are down-regulated along with the adenosine receptor in rat adipocytes.
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Department of Internal Medicine and Pharmacology, University of Texas Medical Branch, Galveston 77550.
Johnson
J L
JL
Milligan
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D000241:Adenosine / Q000031:analogs & derivatives*; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007106:Immune Sera; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis; D010660:Phenylisopropyladenosine / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000187:drug effects / Q000378:metabolism*
D007106:Immune Sera; D009842:Oligopeptides; D011983:Receptors, Purinergic; D009243:NAD; D010660:Phenylisopropyladenosine; D019204:GTP-Binding Proteins; D000241:Adenosine