Stimulation of cAMP Synthesis by Gi-coupled Receptors upon Ablation of Distinct Gαi Protein Expression: Gi SUBTYPE SPECIFICITY OF THE 5-HT1A RECEPTOR

Ya Fang Liu,Mohammad H Ghahremani,Mark M Rasenick,Karl H Jakobs,Paul R Albert

doi:10.1074/jbc.274.23.16444

Abstract

The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level.

Highlights

The three G␣i subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each G␣i antisense rat cDNA construct
Using anti-G␣i subunit antibodies to block receptor coupling, it has been reported that inhibition of AC by ␣2-adrenergic receptors in platelet membranes is mediated by G␣i2 [12] and that 5-HT1A receptor-induced inhibition of cAMP synthesis in HeLa cell membranes is preferentially mediated by G␣i3 [13]
The contemporaneous expression of all three G␣i subunits appeared to be essential for both 5-HT1A and muscarinic M4 receptor-mediated inhibition of unstimulated cAMP synthesis (Table III)

Summary

Introduction

The three G␣i subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each G␣i antisense rat cDNA construct. 5-HT1A and muscarinic M4 receptor may couple dominantly to G␣i1 and G␣i3, respectively, to inhibit cAMP production Upon removal of these G␣i subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve G␤␥-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Using anti-G␣i subunit antibodies to block receptor coupling, it has been reported that inhibition of AC by ␣2-adrenergic receptors in platelet membranes is mediated by G␣i2 [12] and that 5-HT1A receptor-induced inhibition of cAMP synthesis in HeLa cell membranes is preferentially mediated by G␣i3 [13] This approach, is limited to cell-free preparations and depends on the specificity of the antibodies used. We have used expression of antisense constructs to selectively deplete particular G proteins and assessed their contribution to receptor coupling (16 –18)

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Conclusion