Macrophage Class A Scavenger Receptors (SR-A/CD204) mediate distinct functions including macrophage adhesion to modified matrix proteins, clearance of bacterial products, and internalization of modified lipoproteins. These functions of SR-A are associated with the activation of different signaling pathways which differentially regulate SR-A function. The mechanisms segregating SR-A function and regulation are not well defined, but may involve compartmentalization of SR-A with signaling proteins in distinct membrane domains. Lipid rafts are free cholesterol rich regions that regulate the function of many receptors by spatially segregating components of signal transduction, thus providing an environment for localized activation of receptor. Therefore, we hypothesize that SR-A function and coupling to specific signaling pathways is spatially regulated by lipid rafts.
To test this hypothesis, we initially examined SR-A localization in lipid rafts using an optimized detergent free isolation protocol. We found that SR-A localizes in both lipid rafts and non-raft plasma membrane; however, the concentration of SR-A is two-fold higher in lipid rafts than in the non-raft membrane suggesting that the localization of SR-A is regulated. SR-A localized in lipid rafts even when 49 of the 55 amino acids in the cytoplasmic domain were deleted. The ability of the truncated receptor to mediate cell adhesion but not ligand internalization indicates that SR-A localization in lipid rafts is associated with cell adhesion.
To determine the role of lipid rafts in SR-A mediated cell adhesion, we used the cholesterol chelating compound Methyl-beta-cyclodextrin (MβCD) to assess the effect of lipid raft disruption on SR-A mediated cell attachment and activation of signals associated with cell adhesion. We found that cholesterol depletion and disruption of lipid rafts significantly inhibited the SR-A mediated macrophage attachment. This inhibition was not associated with change in SR-A on the cell surface or the ability of SRA to activate the PI3K signaling pathway. Rather, lipid rafts disruption specifically abolished the activation of phospholipase A2 and prostaglandin production during SR-A mediated macrophage adhesion. The ability of exogenously added arachidonic acid to reverse the inhibitory effect of MβCD indicates that SR-A mediated activation of phospholipase A2 requires lipid rafts.
To determine the role of lipid rafts in SR-A mediated ligand internalization, we examined macrophage uptake of the SR-A ligand acetylated LDL (AcLDL) following MβCD treatment. We found that the SR-A mediated uptake of AcLDL, but not native LDL, was reduced by 40% following MβCD treatment. This indicates that AcLDL is taken up in part by a lipid raft dependent process. Because the effect of MβCD was observed in the absence of caveolin, we conclude that the process is independent of caveolae. To attempt to identify this lipid raft-dependent, SR-A mediated ligand uptake pathway, we used pharmacological inhibitors to study AcLDL uptake via phagocytosis and macropinocytosis. My results indicate that the lipid raft dependent SR-A mediated uptake of AcLDL involves macropinocytosis. However, we cannot exclude the possibility that SR-A mediated phagocytosis involves lipid rafts.
Overall, our results demonstrate that SR-A is localized in lipid rafts, and that lipid rafts play an important role in regulating the SR-A mediated function. Lipid rafts specifically couple SR-A to the activation of a phospholipase A2 dependent signaling pathway to promote macrophage adhesion and prostaglandin production. Lipid rafts are also involved in SR-A dependent ligand internalization. Together, our results suggest that altering lipid raft function by increasing or decreasing cholesterol, for example in atherosclerosis or statin treatment, may increase or decrease SR-A function to affect macrophage recruitment/retention, ligand clearance and disease progression.
|Advisor:||Post, Steven R.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 76/01(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Health sciences, Immunology|
|Keywords:||Macrophage Class A Scavenger Receptors, Macrophage adhesion, Modified matrix proteins|
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