Coupling of 5-HT1A autoreceptors to inhibition of mitogen-activated protein kinase activation via G beta gamma subunit signaling

The 5-HT1A receptor is expressed presynaptically as the primary somatodendritic autoreceptor on serotonergic raphe neurons, and postsynaptically in several brain regions. Signaling of the 5-HT1A autoreceptor was studied in RN46A cells, a model of serotonergic raphe neurons that express endogenous 5-HT1A receptors. In undifferentiated RN46A cells stably transfected with the wild-type 5-HT1A receptor, 5-HT1A receptor activation inhibited forskolin-induced cyclic adenosine monophosphate (cAMP) formation (by 50%), increased [Ca2+]i, and induced a novel inhibition (up to 60%) of phospho-p42/p44-mitogen-activated protein kinase (MAPK). Upon differentiation of non-transfected or 5-HT1A-transfected RN46A cells, agonist-mediated inhibition of MAPK was enhanced. These actions were blocked by pretreatment with pertussis toxin indicating mediation via Gi/Go proteins and the calcium response was blocked by preactivation of protein kinase C (PKC). In cells overexpressing the G beta gamma scavenger carboxyl-terminal domain of G protein receptor kinase 2 (GRK-CT), 5-HT1A receptor activation inhibited cAMP formation, but coupling to calcium mobilization and inhibition of MAPK was abolished. The activity of 5-HT1A receptors containing mutations of PKC sites in the second (i2: T149A) or third intracellular loop (i3: T229A/S253G/T343A) was tested. At comparable levels of receptor expression, the signaling of the 5-HT1A i3 mutant was similar to the 5-HT1A wild-type receptor, while the i2 and quadruple (i2/i3) mutants failed to couple to G beta gamma-mediated increase in [Ca2+]i or inhibition of MAPK, but did couple to G alpha i-mediated inhibition of cAMP. Thus, the i2-domain of the 5-HT1A autoreceptor is crucial for coupling to G beta gamma subunits and their subsequent responses (e.g. calcium mobilization and inhibition of MAPK activity).