Short- and long-term regulation of adenylyl cyclase activity by δ-opioid receptor are mediated by Gα12 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 (G alpha(i2), G alpha(i3), and G alpha(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 G alpha(i2), and not the C351L mutants of G alpha(i3) or G alpha(o), rescued the short- term effect of DPDPE after pertussis toxin treatment. The ability of G alpha(i2) in mediating DOR inhibition of adenylyl cyclase activity was also reflected in the ability of G alpha(i2), not G alpha(i3) or G alpha(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 G alpha(i2) restored the adenylyl cyclase superactivation after pertussis toxin treatment. More importantly, the C352L mutant of G alpha i2 remained associated with DOR after long-term agonist and pertussis toxin treatment whereas the wild-type G alpha(i2) did not. These data suggest that G alpha(i2) serves as the signaling molecule in both DOR-mediated short- and long-term regulation of adenylyl cyclase activity.