Regulated Endocytosis of G-protein-coupled Receptors by a Biochemically and Functionally Distinct Subpopulation of Clathrin-coated Pits

Abstract

␤-2 Adrenergic receptors (B2ARs) are endocytosed by clathrin-coated pits. This process serves specialized functions in signal transduction and receptor regulation, raising the question of whether B2ARs are associated with biochemically specialized membrane vesicles during their endocytic trafficking. Here we show that B2ARs are endocytosed by a distinct subpopulation of clathrin-coated pits, which represent a limited subset of coated pits present in the plasma membrane, even in cells overexpressing both B2ARs and ␤-arrestin. Coated pits mediating agonist-induced endocytosis of B2ARs differ from other coated pits mediating constitutive endocytosis of transferrin receptors in their temperature dependence for fission from the plasma membrane and in the association of their membrane coats with ␤-arrestin. Endocytosis of these coated pits generates endocytic vesicles selectively enriched in B2ARs, which fuse within ϳ10 min after their formation with a common population of endosomes containing both B2ARs and transferrin receptors. These observations demonstrate, for the first time, the existence of a functionally and biochemically distinct subpopulation of clathrin-coated pits that mediate the agonist-regulated endocytosis of G-protein-coupled receptors, and they suggest a new model for the formation of compositionally specialized membrane vesicles at the earliest stage of the endocytic pathway.Many G protein-coupled receptors (GPCRs), 1 such as the ␤-2 adrenergic receptor (B2AR), are endocytosed by clathrin-coated pits and follow an endocytic pathway similar to constitutively endocytosed nutrient receptors (such as the transferrin receptor (TfnR)) (1-3). Clathrin-mediated endocytosis delivers both classes of receptor to endosomes, where ligand-receptor dissociation and protein sorting occur (4). However, in addition to these conserved functions of the early endocytic pathway, clathrin-mediated endocytosis serves specialized functions in GPCR regulation and signal transduction. Endocytosis of certain GPCRs, such as the B2AR, promotes dephosphorylation and functional resensitization of receptors following agonistinduced desensitization (5, 6). Additionally, clathrin-mediated endocytosis also plays an important role in promoting receptordependent activation of mitogen-activated protein kinase (MAPK) cascades (7).Endocytosis by clathrin-coated pits serves specialized functions for other types of membrane proteins, such as synaptic vesicle membrane proteins and certain membrane transporters. In these cases, the specialized functions of the endocytic pathway are mediated by the formation of distinct endomembrane vesicles (e.g. synaptic vesicles and Glut4-containing vesicles), which diverge from a common early endosomal intermediate and contain a compositionally refined subset of membrane proteins (8 -11). Accordingly, these considerations raise the question of whether the endocytic trafficking of GPCRs may also involve specialized membranes. However, in contrast to synaptic vesicle membrane proteins, which are sorted after...

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