Lipid rafts constrain basal α1A-adrenergic receptor signaling by maintaining receptor in an inactive conformation

We have reported that the α1A-adrenergic receptor (α1AAR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-β-cyclodextrin (MCD) sequestration of cholesterol affects α1AAR signaling. Unexpectedly, MCD increased α1AAR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface α1AAR, 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 α1AAR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating α1AAR signaling following stimulation.