Abstract
Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes.Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment.Conclusion: These results suggest that different membrane domains contribute to the formation of distinct pools of cAMP under basal conditions as well as following receptor stimulation in adult ventricular myocytes.
Highlights
The diffusible second messenger cAMP mediates responses to a wide array of neurotransmitters, hormones, and autacoids acting through a variety of G protein-coupled receptor (GPCR) in virtually every cell in the human body
We have previously reported that, while there is some loss of cholesterol content, cells maintained in culture under these conditions do not exhibit a marked loss in caveolae at cell surface, and compartmentalized cAMP responses associated with membrane microdomains are preserved (Agarwal et al, 2011; Macdougall et al, 2012)
The non-uniform distribution of signaling proteins between lipid raft and non-raft domains of the plasma membrane is believed to be an important factor contributing to the compartmentation of cAMP signaling in cardiac myocytes (Harvey and Calaghan, 2012)
Summary
The diffusible second messenger cAMP mediates responses to a wide array of neurotransmitters, hormones, and autacoids acting through a variety of GPCRs in virtually every cell in the human body. What is even more remarkable is that even though cAMP regulates multiple processes in any given cell, receptor dependent stimulation of cAMP production often elicits unique downstream responses. Only the cAMP produced by βARs regulates the electrical and mechanical properties of these cells (Buxton and Brunton, 1983; Steinberg and Brunton, 2001). Observations such as this led to the idea that cAMP signaling must be compartmentalized
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