Stimulation of cAMP synthesis by Gi-coupled receptors upon ablation of distinct Galphai protein expression. Gi subtype specificity of the 5-HT1A receptor.
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.
Liu
Y F
YF
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Ghahremani
M H
MH
Rasenick
M M
MM
Jakobs
K H
KH
Albert
P R
PR
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis*; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D037342:Pertussis Toxin; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic / Q000378:metabolism; D011985:Receptors, Serotonin / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D011518:Proto-Oncogene Proteins; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D010566:Virulence Factors, Bordetella; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat; D000262:Adenylyl Cyclases; D002118:Calcium