Short- and long-term regulation of adenylyl cyclase activity by δ-opioid receptor are mediated by Gα_i2 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 α-subunits of G_i/o that are expressed in neuroblastoma Neuro2A cells (Gα_i2, Gα_i3, and Gα_o), we examined the identities of the G proteins involved in the short- and long-term action of the δ-opioid receptor (DOR). Pertussis toxin pretreatment completely abolished the ability of [D-Pen²,D-Pen ⁵]-enkephalin (DPDPE) to inhibit forskolin-stimulated intracellular cAMP production. Expression of the C352L mutant of Gα_i2, and not the C351L mutants of Gα_i3 or Gα_o, rescued the short-term effect of DPDPE after pertussis toxin treatment. The ability of Gα_i2 in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of Gα_i2, not Gα_i3 or Gα_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 Gα_i2 restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of Gα_i2 remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type Gα_i2 did not. These data suggest that Gα_i2 serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.