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Effects of freezing and protein cross-linker on isolating membrane raft-associated proteins.

ABSTRACT: Since the discovery of cellular membrane rafts, the defining of these domains has remained ambiguous due to a great number of isolation procedures proposed for the extraction of the rafts from cells. Characterization of membrane rafts using Triton X-100 insolubility is limited by the fact that weak interactions between proteins and lipids within the membrane rafts cannot be detected. In order to study the role of membrane rafts in cell signal transduction, it is crucial that weak membrane raft-associated proteins are detected. In this report, we demonstrate that by incorporating 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) crosslinking and freezing at -80°C into the membrane raft isolation procedure of HaCaT cells, both membrane raft-associated proteins caveolin-1 and Fas receptor are able to be reproducibly isolated into a single fraction containing the membrane rafts of the cells.

SUBMITTER: George KS

PROVIDER: S-EPMC3122141 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Effects of freezing and protein cross-linker on isolating membrane raft-associated proteins.

George Kimberly Suzanne KS Wu Qiong Q Wu Shiyong S

BioTechniques 20101101 5

Since the discovery of cellular membrane rafts, the defining of these domains has remained ambiguous due to a great number of isolation procedures proposed for the extraction of the rafts from cells. Characterization of membrane rafts using Triton X-100 insolubility is limited by the fact that weak interactions between proteins and lipids within the membrane rafts cannot be detected. In order to study the role of membrane rafts in cell signal transduction, it is crucial that weak membrane raft-a ...[more]

PMID: 21091450

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Project description: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 ?-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.

Dataset Information

Effects of freezing and protein cross-linker on isolating membrane raft-associated proteins.

Publications

Effects of freezing and protein cross-linker on isolating membrane raft-associated proteins.

Similar Datasets

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