Studies directed towards the plasma membrane have provided evidence for the existence of submicrometre cholesterol-rich domains, termed lipid rafts. These specialised domains of the plasma membrane are centrally involved in the containment of macromolecular signalling complexes and evidence suggests that Ca²⁺-sensitive adenylyl cyclases are resident in rafts. This restrictive localisation is critical for the regulation of Ca²⁺-stimulable AC8 (Smith et al. 2002) and Ca²⁺-inhibitable AC6 by capacitative calcium entry (Fagan et al. 2000). Interestingly however, Ca²⁺-insensitive adenylyl cyclases appear to be excluded from lipid rafts. The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remains unknown. To address this question, a series of myc-tagged chimaeras were produced consisting of Ca²⁺-inhibitable AC5 with major domains exchanged exchanged major domains substituted from Ca²⁺-insensitive AC7. The AC5-AC7 chimaeras were expressed in HEK 293 cells and lipid rafts were isolated by a non-detergent based fractionation method. Confocal imaging was used to investigate plasma membrane targeting. Demonstrating the separation of rafts from bulk membrane, Western blotting revealed an enrichment of the raft marker proteins caveolin and flotillin in the buoyant membrane fraction. Wild type AC5 was enriched in the buoyant membrane fraction, whereas wild-type AC7 was only detectable in the bulk membrane fraction. Confocal imaging demonstrated that both AC5 and AC7 were plasma membrane bound. Exchanging the N-terminus or the transmembrane domains of AC5 with similar regions from AC7 did not affect raft targeting or Ca²⁺-inhibition in vitro. Exchanging the C1b domain of AC5 produced an ER retained construct and an intact C2b domain was essential for correct protein handling at the level of the ER for AC5 but not AC7. Therefore the C1b and C2b domains are essential for enabling restrictive localisation of AC5 to lipid raft microdomains.

This work was supported by the Wellcome Trust