Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ subunits exert opposing effects depending on the G-protein source.
Direct phosphorylation of AC2 (adenylyl cyclase 2) by PKC (protein kinase C) affords an opportunity for AC2 to integrate signals from non-canonical pathways to produce the second messenger, cyclic AMP. The present study shows that stimulation of AC2 by pharmacological activation of PKC or muscarinic receptor activation is primarily the result of phosphorylation of Ser490 and Ser543, as opposed to the previously proposed Thr1057. A double phosphorylation-deficient mutant (S490/543A) of AC2 was insensitive to PMA (phorbol myristic acid) and CCh (carbachol) stimulation, whereas a double phosphomimetic mutant (S490/543D) mimicked the activity of PKC-activated AC2. Putative Gβγ-interacting sites are in the immediate environment of these PKC phosphorylation sites (Ser490 and Ser543) that are located within the C1b domain of AC2, suggesting a significant regulatory importance of this domain. Consequently, we examined the effect of both Gq-coupled muscarinic and Gi-coupled somatostatin receptors. Employing pharmacological and FRET (fluorescence resonance energy transfer)-based real-time single cell imaging approaches, we found that Gβγ released from the Gq-coupled muscarinic receptor or Gi-coupled somatostatin receptors exert inhibitory or stimulatory effects respectively. These results underline the sophisticated regulatory capacities of AC2, in not only being subject to regulation by PKC, but also and in an opposite manner to Gβγ subunits, depending on their source.
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
Jia X
JX
Shen
Sebastian
S
Wachten
Michelle L
ML
Halls
Katy L
KL
Everett
Dermot M F
DM
Cooper
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D017481:Receptors, Somatostatin / Q000378:metabolism; D059010:Single-Cell Analysis
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D043587:Receptor, Muscarinic M3; D017481:Receptors, Somatostatin; D002217:Carbachol; D000242:Cyclic AMP; D011493:Protein Kinase C; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000262:Adenylyl Cyclases; C100622:adenylyl cyclase 2