A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.

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The ␤ 2 -adrenergic receptor and ␦ opioid receptor represent distinct G protein-coupled receptors that undergo agonist-induced endocytosis via clathrin-coated pits but differ significantly in their postendocytic sorting between recycling and degradative membrane pathways, respectively. Previous results indicate that a distal portion of the carboxyl-terminal cytoplasmic domain of the ␤ 2 -adrenergic receptor, which engages in PDZ domain-mediated protein interaction, is required for efficient recycling of receptors after agonist-induced endocytosis. Here we demonstrate that a four-residue sequence (DSLL) comprising the core of this protein interaction domain functions as a transplantable endocytic sorting signal that is sufficient to re-route endocytosed ␦ opioid receptor into a rapid recycling pathway, to inhibit proteolytic down-regulation of receptors, and to mediate receptor-autonomous sorting of mutant receptors from the wild type allele when co-expressed in the same cells. These observations define a transplantable signal mediating rapid recycling of a heterologous G protein-coupled receptor, and they suggest that rapid recycling of certain membrane proteins does not occur by bulk membrane flow but is instead mediated by a specific endocytic sorting mechanism.

␤-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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Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca(2+) on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va.

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (ϳ1:24 Myosin-Va:tubulin; K d ‫؍‬ 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca 2؉ , ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 M Ca 2؉ , ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments. INTRODUCTIONMyosin-Va (Myo5a) is the best characterized of three class V myosins identified in vertebrates (Berg et al., 2001). Myo5a is a two-headed motor, consisting of two heavy chains and multiple light chains (reviewed in Reck-Peterson et al., 2000). The N-terminal half of each heavy chain consists of a head or motor domain followed by a neck domain composed of six IQ motifs, each of which binds a calmodulin light chain although chicken Myo5a also contains a pair of myosin-II essential light chains . The C-terminal tail domain consists of a proximal dimerization stalk of largely coiled-coil forming alpha helix followed by a globular domain that has been shown to interact with numerous presumed "cargo" molecules (Reck-Peterson et al., 2000;Karcher et al., 2002;Langford, 2002). The tail domain also contains at least a pair of the low-molecular-weight light chain, LC8 or PIN, first identified as a light chain of the microtuble (MT) motor, dynein, but subsequently shown to interact with numerous other proteins . Biochemical studies on both tissue-purified and baculovirus-expressed Myo5a (reviewed in Reck-Peterson et al., 2000;Mehta, 2001) have shown that it is a Ca 2ϩ -regulated, barbed-end directed, processive motor that takes large, ϳ36-nm steps in a hand-overhand manner (Forkey et al., 2003; Yildiz et al., 2003) along the actin filament.Insights into the potential functions for Myo5a have come from several lines of investigation, including identification of organelles and proteins that interact with Myo5a (ReckPeterson et al., 2000;Karcher et al., 2002;Langford, 2002). The most powerful strategy has been the phenotypic characterization of lethal alleles of the dilute (Myo5a lethal mutant) mouse and cells derived from them. The dilute mice develop severe seizures and die within 3 wk after birth. The primary neuronal defect thought to be responsible is the absence of smooth endoplasmic reticulum (SER) within the dendritic spines of Purkinje neurons (Takagishi et al., 1996). Anal...

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