Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation.
Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of Gi protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP3. By monitoring cAMP levels in real time by means of Förster resonance energy transfer (FRET)-based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP3-dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP3 that leads to pronounced alterations of cytosolic cAMP levels.
*Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
Gopireddy Raghavender
GR
Reddy
*Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
Hariharan
H
Subramanian
*Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
Alexandra
A
Birk
*Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
Markus
M
Milde
*Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.
Viacheslav O
VO
Nikolaev
*Faculty of Pharmacy, Philipps University, Marburg, Marburg, Germany; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany moritz.buenemann@staff.uni-marburg.de.
Moritz
M
Bünemann
D000262:Adenylyl Cyclases / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6; C419510:adenylyl cyclase type V
FRET; GPCR; Gαi; PI3K; cAMP