C1 and C2 Domains of Human Adenylyl Cyclase 6 are Targeted to Lipid Rafts and Caveolae

Abstract

Previous data has shown that adenylyl cyclase type 6 (AC6) is enriched in lipid rafts or caveolae of various cell types but that certain other isoforms of AC are excluded from these domains. The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown. In this study, we constructed proteins corresponding to the four intracellular domains of human AC6: the N-terminus, C1, C2 and the C-terminus. We transfected each protein in Cos-7 cells, which express caveolin-1, and HEK-293 cells, which lack prototypical caveolins, and assessed their localization. Lipid rafts and caveolae were isolated from plasma membranes using a non-detergent fractionation method and fractions were analyzed by immunoblotting. We also used co-immunopreciptation, Triton-X-100 insolubility and immunoflurescence microscopy to identify the subcellular targeting of each expressed intracellular domain protein. We found that the N-terminal and C-terminal domains of AC6 are not associated with lipid rafts or raft-localized proteins. By contrast, the C1 domain was strongly localized in lipid rafts and the C2 domain was more weakly localized in lipid rafts, suggesting that these portions of AC6 are integral to its localization in these domains. There were no differences in C1 or C2 localization patterns between Cos-7 and HEK-293 cells, implying that the interaction with caveolins is not important for the localization of AC6. Thus, the structural elements of the AC6 protein responsible for its localization in lipid rafts and caveolae appear to be contained primarily in the C1 but also in the C2 domains. Supported by AHA grant 0555291B.