Short- and long-term regulation of adenylyl cyclase activity by delta-opioid receptor are mediated by Galphai2 in neuroblastoma N2A cells.
Activation of the opioid receptor results in short-term inhibition of intracellular cAMP levels followed by receptor desensitization and subsequent increase of cAMP above the control level (adenylyl cyclase superactivation). Using adenovirus to deliver pertussis toxin-insensitive mutants of the alpha-subunits of G(i/o) that are expressed in neuroblastoma Neuro2A cells (Galpha(i2), Galpha(i3), and Galpha(o)), we examined the identities of the G proteins involved in the short- and long-term action of the delta-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [d-Pen(2), d-Pen(5)]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Galpha(i2), and not the C351L mutants of Galpha(i3) or Galpha(o), rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Galpha(i2) in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Galpha(i2), not Galpha(i3) or Galpha(o), to coimmunoprecipitate with DOR. Coincidently, after long-term DPDPE treatment, pertussis toxin treatment eliminated the antagonist naloxone-induced superactivation of adenylyl cyclase activity. Again, only the C352L mutant of Galpha(i2) restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Galpha(i2) remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Galpha(i2) did not. These data suggest that Galpha(i2) serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.
Zhang
Lei
L
Department of Pharmacology, Medical School, University of Minnesota, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA. zhang247@umn.edu
Tetrault
Joan
J
Wang
Wei
W
Loh
Horace H
HH
Law
Ping-Yee
PY
D000256:Adenoviridae / Q000235:genetics; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000037:antagonists & inhibitors / Q000494:pharmacology; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D009270:Naloxone / Q000494:pharmacology; D009447:Neuroblastoma; D037342:Pertussis Toxin / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000067956:Adenylyl Cyclase Inhibitors; D017465:Receptors, Opioid, delta; D009270:Naloxone; D020881:Enkephalin, D-Penicillamine (2,5)-; D000242:Cyclic AMP; D037342:Pertussis Toxin; D051960:GTP-Binding Protein alpha Subunit, Gi2; D000262:Adenylyl Cyclases