Lipid rafts constrain basal alpha(1A)-adrenergic receptor signaling by maintaining receptor in an inactive conformation.
We have reported that the alpha(1A)-adrenergic receptor (alpha(1A)AR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-beta-cyclodextrin (MCD) sequestration of cholesterol affects alpha(1A)AR signaling. Unexpectedly, MCD increased alpha(1A)AR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface alpha(1A)AR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal alpha(1A)AR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating alpha(1A)AR signaling following stimulation.
Lei
Beilei
B
Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA. lei00002@mc.duke.edu
Morris
Daniel P
DP
Smith
Michael P
MP
Schwinn
Debra A
DA
D000818:Animals; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C547713:ADRA1A protein, human; C547715:Adra1a protein, rat; D007295:Inositol Phosphates; D018340:Receptors, Adrenergic, alpha-1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D048051:p38 Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins