Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates.
We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.
McKenzie
F R
FR
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Kelly
E C
EC
Unson
C G
CG
Spiegel
A M
AM
Milligan
G
G
D000244:Adenosine Diphosphate / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D001769:Blood / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000276:immunology*; D037342:Pertussis Toxin; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011957:Receptors, Opioid / Q000378:metabolism; D017465:Receptors, Opioid, delta; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000906:Antibodies; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D010566:Virulence Factors, Bordetella; D004743:Enkephalin, Leucine; D000244:Adenosine Diphosphate; D016308:Enkephalin, Leucine-2-Alanine; D037342:Pertussis Toxin; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases