Received for publication October 19, 2004.
Revised December 21, 2004.
Accepted for publication December 21, 2004.

-arrestin Dependent Spontaneous _1a-Adrenoceptor Endocytosis causes Intracellular Transportation of -blockers via Recycling Compartments

Abstract

The antagonist ligand BODIPY-FL-prazosin (QAPB) fluoresces when bound to bovine _1a-adrenoceptors (ARs). Data indicates that the receptor-ligand complex is spontaneously internalized by -arrestin-dependent endocytosis. Internalization of the ligand did not occur in -arrestin-deficient cells, was blocked or reversed by another ₁ ligand, phentolamine, indicating it to reflect binding to the orthosteric recognition site, and was prevented by blocking clathrin-mediated endocytosis. The ligand showed rapid, diffuse, low-intensity, surface binding, superseded by punctate intracellular binding that developed to equilibrium in 50-60 min, and was reversible on ligand removal, indicating a dynamic equilibrium. In cells expressing a human _1a-AR-EGFP2 fusion protein, BODIPY-R-558/568-prazosin (RQAPB) co-localized with the fusion, indicating that the ligand gained access to all compartments containing the receptor and, conversely, that the receptor has affinity for the ligand at all of these sites. The distribution of QAPB binding sites was similar for receptors with or without EGFP2, validating the fusion protein as an indicator of receptor location. The ligand partially co-localized with -arrestin in recycling and late endosomes indicating receptor transit without destruction. Organelles containing receptors showed considerable movement consistent with a transportation function. This was absent in -arrestin-deficient cells indicating that both constitutive receptor internalization and subsequent intracellular transportation are -arrestin-dependent. Calculations of relative receptor number suggest that at steady state less than 30% of receptors reside on the cell surface and that recyling is extremely rapid. We conclude that _1a-ARs recycle rapidly by an agonist-independent, constitutive, -arrestin-dependent process and that this can transport "-blockers" into cells carrying these receptors.

Key words: Adrenergic, Receptor synthesis/trafficking, Sequestration/Internalization, GRKs, barrestins, Recycling, Fluorescence techniques