title: Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase.
abstract: Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca(2+) entry (CCE) over other modes of Ca(2+) increase. To directly address the possibility that these ACs reside in discrete Ca(2+) microdomains, we tethered a Ca(2+) sensor, GCaMP2, to the N-terminus of Ca(2+)-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca(2+)-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca(2+) rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca(2+) fluxes during Ca(2+) mobilization and non-specific Ca(2+) entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca(2+)-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca(2+) signals seen throughout the cytosol. By contrast, a unique Ca(2+) microdomain surrounds AC8 that promotes selectivity for Ca(2+) signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca(2+) signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.
authors.affiliation: Department of Pharmacology, Tennis Court Road, University of Cambridge, CB2 1PD, UK.
authors.forename: Debbie
authors.initials: D
authors.lastname: Willoughby
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wachten
authors.forename: Nanako
authors.initials: N
authors.lastname: Masada
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics
chemical_list: D015335:Molecular Probes; D020033:Protein Isoforms; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C100622:adenylyl cyclase 2; C118292:adenylyl cyclase 8; D002118:Calcium