The Proximal Segments of the Cytosolic Domain of Adenylyl Cyclase Type 6 Localize to Plasma Membrane Lipid Rafts and Caveolae

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Lipid rafts, rich in cholesterol and sphingolipids, display a high concentration of signaling proteins such as receptors, G-proteins, and effectors molecules including certain isoforms of adenylyl cyclases (AC). The mechanism by which AC6 is localized to lipid rafts or caveolae is unknown, but previous work indicates a role for the C1 domain. In this study, we constructed recombinant proteins that comprise the four segments of the C1 domain of human AC6. The proteins correspond to amino acids 306–387, 388–490, 491–589 and 590–692 of human AC6. We transfected each protein in cos-7 cells, which express caveolin-1, and assessed their localization via a fused GFP. Lipid rafts and caveolae were isolated from plasma membrane using a detergent-based fractionation method and fractions were analyzed by immunoblotting. We also used co-immunoprecipitation and immunoflurescence microscopy to identify the subcellular targeting to each expressed C1 domain fragment. We found that only proteins 306 and 388 were associated with lipid rafts, suggesting that these portions of AC6 play a role in its localization. Thus, certain structural elements responsible for AC6 localization in lipid rafts and caveolae appear to be contained in the proximal portion of the C1 domain.


This abstract was originally published in FASEB Journal, volume 21, issue 6, in 2007.


Federation of American Society of Experimental Biology (FASEB)