Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells.
Microdomains have been proposed to explain specificity in the myriad of possible cellular targets of cAMP. Local differences in cAMP levels can be generated by phosphodiesterases, which control the diffusion of cAMP. Here, we address the possibility that adenylyl cyclases, the source of cAMP, can be primary architects of such microdomains. Distinctly regulated adenylyl cyclases often contribute to total cAMP levels in endogenous cellular settings, making it virtually impossible to determine the contribution of a specific isoform. To investigate cAMP dynamics with high precision at the single-isoform level, we developed a targeted version of Epac2-camps, a cAMP sensor, in which the sensor was tagged to a catalytically inactive version of the Ca(2+)-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca(2+) in GH(3)B(6) pituitary cells. Ca(2+) release triggered by thyrotropin-releasing hormone stimulated the minor endogenous AC8 species. cAMP levels were decreased by inhibition of AC5 and AC6, and simultaneous activation of phosphodiesterases, in different compartments of the same cell. These findings demonstrate the existence of distinct adenylyl-cyclase-centered cAMP microdomains in live cells and open the door to their molecular micro-dissection.
Wachten
Sebastian
S
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, England, UK.
Masada
Nanako
N
Ayling
Laura-Jo
LJ
Ciruela
Antonio
A
Nikolaev
Viacheslav O
VO
Lohse
Martin J
MJ
Cooper
Dermot M F
DM
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000166:cytology; D015202:Protein Engineering; D020033:Protein Isoforms / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D015335:Molecular Probes; D020033:Protein Isoforms; C442864:RAPGEF4 protein, human; D013973:Thyrotropin-Releasing Hormone; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D000262:Adenylyl Cyclases