Article,
Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells
J Cell Sci, 123 (Pt 1): 95-106 (January 2010)Wachten, Sebastian Masada, Nanako Ayling, Laura-Jo Ciruela, Antonio Nikolaev, Viacheslav O Lohse, Martin J Cooper, Dermot M F RG 31760/Wellcome Trust/United Kingdom Research Support, Non-U.S. Gov't England Journal of cell science J Cell Sci. 2010 Jan 1;123(Pt 1):95-106..
Abstract
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.
