The alpha1a-adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin-coated pits

The alpha(1a)-adrenergic receptor (alpha(1a)AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha(1a)AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha(1a)AR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-beta-cyclodextrin eliminates these light rafts. To confirm the presence of the alpha(1a)AR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha(1a)AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha(1a)AR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha(1a)AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha(1a)AR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-beta-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-alpha(1a)AR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha(1a)AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.