The α1a-Adrenergic Receptor Occupies Membrane Rafts with Its G Protein Effectors but Internalizes via Clathrin-coated Pits

• Published in: The Journal of biological chemistry 2008-02, Vol.283 (5), p.2973
• Main Authors: Daniel P. Morris, Beilei Lei, Yue-Xuan Wu, Gregory A. Michelotti, Debra A. Schwinn
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M705795200

The α 1a -adrenergic receptor (α 1a AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the α 1a AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal α 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-β-cyclodextrin eliminates these light rafts. To confirm the presence of the α 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 α 1a AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the α 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 α 1a AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface α 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-β-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-α 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 α 1a AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.