title: Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu.
abstract: The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD,  UK.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Ayling
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Halls
authors.forename: Gerald R V
authors.initials: GR
authors.lastname: Hammond
authors.forename: Luis
authors.initials: L
authors.lastname: Vaca
authors.forename: Jonathan
authors.initials: J
authors.lastname: Pacheco
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D036681:Fluorescence Recovery After Photobleaching; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
chemical_list: D002784:Cholesterol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8