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The non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, beta(2)-adaptin (the beta-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and beta(2)-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated beta(2)-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in beta(2)-adaptin binding and LIELD (residues 376-380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to beta(2)-adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-DeltaLIELD) or beta(2)-adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the beta(2)-adrenergic receptor, while arrestin/beta(2)-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and beta(2)-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated beta(2)-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking.

Arrestins play an important role in regulating the function of G protein-coupled receptors including receptor desensitization, internalization, down-regulation, and signaling via nonreceptor tyrosine kinases and mitogen-activated protein kinases. Previous studies have revealed that arrestins themselves are also subject to regulation. In the present study, we focused on identifying potential mechanisms involved in regulating the function of arrestin-3. Using metabolic labeling, phosphoamino acid analysis, and mutagenesis studies, we found that arrestin-3 is constitutively phosphorylated at Thr-382 and becomes dephosphorylated upon beta(2)-adrenergic receptor activation in COS-1 cells. Casein kinase II (CKII) appears to be the major kinase mediating arrestin-3 phosphorylation, since 1) Thr-382 is contained within a canonical consensus sequence for CKII phosphorylation and 2) wild type arrestin-3 but not a T382A mutant is phosphorylated by CKII in vitro. Functional analysis reveals that mutants mimicking the phosphorylated (T382E) and dephosphorylated (T382A or T382V) states of arrestin-3 promote beta(2)-adrenergic receptor internalization and bind clathrin, beta-adaptin, and Src to comparable levels as wild type arrestin-3. This suggests that the phosphorylation of arrestin-3 does not directly regulate interaction with endocytic (clathrin, beta-adaptin) or signaling (Src) components and is in contrast to arrestin-2, where phosphorylation appears to regulate interaction with clathrin and Src. However, additional analysis reveals that arrestin-3 phosphorylation may regulate formation of a large arrestin-3-containing protein complex. Differences between the regulatory roles of arrestin-2 and -3 phosphorylation may contribute to the different cellular functions of these proteins in G protein-coupled receptor signaling and regulation.

beta-Arrestins, proteins involved in the turn-off of G protein-coupled receptor (GPCR) activation, bind to the beta(2)-adaptin subunit of the clathrin adaptor AP-2. The interaction of beta(2)-adaptin with beta-arrestin involves critical arginine residues in the C-terminal domain of beta-arrestin and plays an important role in initiating clathrin-mediated endocytosis of the beta(2)-adrenergic receptor (beta(2)AR) (Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S., and Caron, M. G. (2000) J. Biol. Chem. 275, 23120--23126). However, the beta-arrestin-binding site in beta(2)-adaptin has not been identified, and little is known about the role of beta-arrestin/AP-2 interaction in the endocytosis of other GPCRs. Using in vitro binding assays, we have identified two glutamate residues (Glu-849 and Glu-902) in beta(2)-adaptin that are important in beta-arrestin binding. These residues are located in the platform subdomain of the C terminus of beta(2)-adaptin, where accessory/adapter endocytic proteins for other classes of receptors interact, distinct from the main site where clathrin interacts. The functional significance of the beta-arrestin/AP-2/clathrin complex in the endocytosis of GPCRs such as the beta(2)AR and vasopressin type II receptor was evaluated using mutant constructs of the beta(2)-adaptin C terminus containing either the clathrin and the beta-arrestin binding domains or the beta-arrestin-binding domain alone. When expressed in human embryonic kidney 293 cells, both constructs acted as dominant negatives inhibiting the agonist-induced internalization of the beta(2)AR and the vasopressin type II receptor. In addition, although the beta(2)-adaptin construct containing both the clathrin and beta-arrestin binding domains was able to block the endocytosis of transferrin receptors, a beta(2)-adaptin construct capable of associating with beta-arrestin but lacking its high affinity clathrin interaction did not interfere with transferrin receptor endocytosis. These results suggest that the interaction of beta-arrestin with beta(2)-adaptin represents a selective endocytic trigger for several members of the GPCR family.

Recent studies have described a widely expressed adaptor-like complex, named AP-3, which is likely involved in protein sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of the subunits of this complex, which we call beta3A-adaptin. The predicted amino acid sequence of beta3A-adaptin reveals that the protein is closely related to the neuron-specific protein beta-NAP (61% overall identity) and more distantly related to the beta1- and beta2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that beta3A-adaptin has a domain organization similar to beta-NAP and to beta1- and beta2-adaptins. beta3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that beta3A-adaptin corresponds to the approximately 140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being delta-adaptin, p47A (now called mu3A) and sigma3 (A or B). beta3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. beta3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of beta3A-adaptin reported here lend support to the idea that AP-3 is a structural and functional homolog of the clathrin-associated adaptors AP-1 and AP-2.

The AP-1 adaptor complex is recruited from the cytosol onto the trans-Golgi network membrane, where it co-assembles with clathrin into a coat that drives vesicle budding. The GTPase ARF1 has been shown to be required for AP-1 recruitment, and here we demonstrate that we can reconstitute full GTPgammaS-dependent recruitment of adaptors onto an enriched trans-Golgi network membrane fraction by adding purified AP-1 and recombinant myristylated ARF1, indicating that these are the only soluble proteins required for binding. To identify some of the membrane proteins involved in recruitment, we have incubated permeabilized metabolically labeled cells with cytosol under conditions that promote adaptor binding, then cross-linked the samples with 3,3'dithiobis(sulfosuccinimidylproprionate), denatured by boiling in SDS, and immunoprecipitated with antibodies against the various subunits. Under these conditions, the adaptor subunits co-precipitate not only with each other and with clathrin, but also with three novel proteins: p75, which is specifically cross-linked to gamma-adaptin; p80, which is specifically cross-linked to beta'-adaptin; and p60, which is specifically cross-linked to AP47. These proteins are all candidates for components of the adaptor docking site on the trans-Golgi network membrane.

The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.

The complete sequence of the beta adaptin subunit of the plasma membrane adaptor complex from coated vesicles has been elucidated. Complementary cDNA clones from human fibroblasts, rat lymphocytes, and bovine lymphocytes have been isolated, sequenced, and compared with each other and with beta adaptin sequences from rat brain (Kirchhausen, T., Nathanson, K.L., Matsui, W., Vaisberg, A., Chow, E.P., Burne, C., Keen, J.H., and Davis, A.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2612-2616). Surprisingly, the 937-amino acid beta adaptin polypeptide is totally conserved between species. This remarkable homology contrasts with the absence of significant sequence similarity between the alpha (Robinson, M.S. (1989) J. Cell Biol. 108, 833-842) and beta adaptins of the plasma membrane adaptor complex. Diversity within each adaptin family is created by the expression of different genes and by tissue-specific differential splicing. The structures of the beta and alpha adaptins can both be divided into two globular domains interconnected by a variable and potentially flexible stalk domain.