U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation.

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.

Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.

Signaling and phosphorylation-impaired mutants of the rat follitropin receptor reveal an activation- and phosphorylation-independent but arrestin-dependent pathway for internalization.

We have previously shown that the rat follitropin receptor (rFSHR) expressed in transfected cells becomes phosphorylated upon stimulation of the cells with agonist or a phorbol ester. Peptide mapping and mutagenesis studies have also shown that the agonist- or phorbol ester-induced phosphorylation of the rFSHR maps to Ser/Thr residues present in the first and third intracellular loops. The experiments presented herein were initially designed to test for the presence of additional phosphorylation sites on the second intracellular loop of the rFSHR. Analysis of two new mutants in which the two threonines in the second intracellular loop (rFSHR-2L) or the two threonines in the second intracellular loop and the seven Ser/Thr residues in the third intracellular loop (rFSHR-2L + 3L) were mutated showed that one or more of the two threonines in the second intracellular loop are phosphorylated in response to phorbol ester, but not in response to agonist stimulation. Since rFSHR-2L and rFSHR-2L + 3L displayed a reduction in agonist-induced signaling, two additional mutants (rFSHR-D389N and rFSHR-Y530F) were constructed in an attempt to better understand the relationship between the agonist-induced activation, phosphorylation, and internalization of the rFSHR. These point mutations impaired agonist-stimulated signal transduction and abolished agonist-induced phosphorylation. Co-transfection studies revealed that the phosphorylation of these mutants can be rescued by overexpression of G protein-coupled receptor kinase 2, but this increased phosphorylation only rescues the internalization of rFSHR-D389N. The internalization of both mutants could be rescued by overexpression of arrestin-3, however. Taken together, these results argue that the agonist-induced activation and phosphorylation of the rFSHR are not essential for internalization. while the interaction of the rFSHR with a nonvisual arrestin is essential for internalization.

Mutation of individual serine residues in the C-terminal tail of the lutropin/choriogonadotropin receptor reveal distinct structural requirements for agonist-induced uncoupling and agonist-induced internalization.

We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20-40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.

The role of receptor kinases and arrestins in G protein-coupled receptor regulation.

G protein-coupled receptors (GPRs) play a key role in controlling hormonal regulation of numerous second-messenger pathways. However, following agonist activation, most GPRs rapidly lose their ability to respond to hormone. For many GPRs, this process, commonly referred to as desensitization, appears to be primarily mediated by two protein families: G protein-coupled receptor kinases (GRKs) and arrestins. GRKs specifically bind to the agonist-occupied receptor, thereby promoting receptor phosphorylation, which in turn leads to arrestin binding. Arrestin binding precludes receptor/G protein interaction leading to functional desensitization. Many GPRs are then removed from the plasma membrane via clathrin-mediated endocytosis. Recent studies have implicated endocytosis in the resensitization of GPRs and have linked both GRKs and arrestins to this process. In this review, we discuss the role of GRKs and arrestins in regulating agonist-specific signaling and trafficking of GPRs.

Mechanism of quenching of phototransduction. Binding competition between arrestin and transducin for phosphorhodopsin.

Quenching of phototransduction in retinal rod cells involves phosphorylation of photoactivated rhodopsin by the enzyme rhodopsin kinase followed by binding of the protein arrestin. Although it has been proposed that the mechanism of arrestin quenching of visual transduction is via steric exclusion of transducin binding to phosphorylated light-activated rhodopsin (P-Rh*), direct evidence for this mechanism is lacking. In this study, we investigated both the role of rhodopsin phosphorylation in modulating its interaction with arrestin and transducin and the proposed binding competition between arrestin and transducin for P-Rh*. While the beta-adrenergic receptor kinase promotes significant arrestin binding to rhodopsin at a phosphorylation stoichiometry of >/=2 mol/mol, rhodopsin kinase promotes arrestin binding at a stoichiometry of approximately 0.9 mol/mol. Moreover, while beta-adrenergic receptor kinase phosphorylation of rhodopsin only modestly decreases transducin binding and activation, rhodopsin kinase phosphorylation of rhodopsin significantly decreases transducin binding and activation. Finally, arrestin competes effectively with transducin for binding to P-Rh* (50% inhibition at approximately 1:1 molar ratio of arrestin:transducin) but has no effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh*), paralleling the functional inhibition by arrestin on P-Rh*-stimulated transducin activation (50% inhibition at approximately 1.7:1 molar ratio of arrestin:transducin). These results demonstrate that a major role of rhodopsin phosphorylation is to promote high-affinity arrestin binding and decrease transducin binding thus allowing arrestin to effectively compete with transducin for binding to photoactivated rhodopsin.

Interaction of arrestins with intracellular domains of muscarinic and alpha2-adrenergic receptors.

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208-Arg387), M3-MR (Gly308-Leu497), or alpha2A/D-adrenergic receptor (AR) (Lys224-Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3 fusion protein, but not to the control GST peptide or i3 peptides derived from the alpha2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified beta-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]beta-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vitro translated [3H]beta-arrestin, [3H]visual arrestin, and [3H]beta-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of beta-arrestin with the M3-MR peptide required both the amino (Gly308-Leu368) and carboxyl portions (Lys425-Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.

Arrestin/clathrin interaction. Localization of the clathrin binding domain of nonvisual arrestins to the carboxy terminus.

We have recently demonstrated that the nonvisual arrestins, beta-arrestin and arrestin3, interact with high affinity and stoichiometrically with clathrin, and we postulated that this interaction mediates internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this study, we localized the clathrin binding domain of arrestin3 using a variety of approaches. Truncation mutagenesis demonstrated that the COOH-terminal half of arrestin3 is required for clathrin interaction. Assessment of the clathrin binding properties of various glutathione S-transferase-arrestin3 fusion proteins indicated that the predominant clathrin binding domain is contained within residues 367-385. Alanine scanning mutagenesis further localized this domain to residues 371-379, and site-directed mutagenesis demonstrated the critical importance of both hydrophobic (Leu-373, Ile-374, and Phe-376) and acidic (Glu-375 and Glu-377) residues in the arrestin3/clathrin interaction. These results are complementary to the observation that hydrophobic and basic residues in clathrin are critical for its interaction with nonvisual arrestins (Goodman, O. B. , Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997) J. Biol. Chem. 272, 15017-15022). Lastly, an arrestin3 mutant in which Leu-373, Ile-374, and Phe-376 were mutated to Ala was significantly defective in its ability to promote beta2-adrenergic receptor internalization in COS-1 cells when compared with wild-type arrestin3. Taken together, these results implicate a discrete region of arrestin3 in high affinity binding to clathrin, an interaction critical for agonist-promoted internalization of the beta2-adrenergic receptor.

Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain.

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.

Modulation of the arrestin-clathrin interaction in cells. Characterization of beta-arrestin dominant-negative mutants.

We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote beta2-adrenergic receptor (beta2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) to function as a dominant-negative with respect to beta2AR internalization and compared its properties with those of beta-arrestin and beta-arrestin-V53D, a previously reported dominant-negative mutant. In vitro studies demonstrated that beta-arrestin-V53D bound better to clathrin than beta-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas beta-arrestin (319-418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the beta2AR in HEK293 cells, beta-arrestin (319-418) effectively inhibited agonist-promoted receptor internalization, whereas beta-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of beta2AR internalization by beta-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both beta-arrestin (319-418) and beta-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of beta-arrestin (319-418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking.

Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor.

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

Globo-series carbohydrate antigens are expressed in different forms on human and murine teratocarcinoma-derived cells.

The glycolipids of human teratocarcinoma-derived cell line NCCIT were compared with those of 5 murine teratocarcinoma-derived cell lines. Glycolipid antigens were identified by cell surface immunofluorescence and high-performance thin-layer chromatography (HPTLC) immunostaining with a panel of monoclonal anti-carbohydrate antibodies. Human NCCIT embryonal carcinoma (EC) cells contained extended globo-series glycolipids Gb5 (galactosyl globoside) and GL7 (sialyl galactosyl globoside) recognized by antibodies to stage-specific embryonic antigens 3 and 4 (SSEA-3 and -4). SSEA-4 was not detected by immunofluorescence on the surface of any of the 5 murine teratocarcinoma-derived cell lines examined; however, SSEA-3 was detected on the surface of murine cell lines resembling primitive endoderm (JC44, NF-PE) and trophectoderm (E6496D). HPTLC analysis revealed a large amount of globoside (Gb4) in these differentiated cells, which may account for their labeling with anti-SSEA-3 antibody. Globo-series glycolipids were also detected in murine EC cells; however, differences were noted between the 2 cell lines examined. F9 cells contained primarily Gb4 and Forssman glycolipid, whereas NF-1 cells contained only minor amounts of Gb4 and lacked Forssman glycolipid entirely. Our results, coupled with the known distribution of Forssman antigen in the egg cylinder-stage mouse embryo, suggest that F9 and NF-1 murine EC cells are replicas of cells at different stages of development of the embryonic ectoderm. Glycolipids of normal mouse embryos were examined for comparison. Gb4 and Forssman glycolipid were presents in both embryonic and extra-embryonic tissues, whereas Gb5 and GL7 were restricted to visceral yolk sac and placenta. Our results demonstrate that human and murine teratocarcinoma-derived cells both synthesize extended globo-series glycolipids; however, oligosaccharide chain elongation takes different pathways in the 2 species. These differences reflect species-related and cell type-specific patterns of glycosylation.

Arrestin-rhodopsin interaction. Multi-site binding delineated by peptide inhibition.

Visual arrestin modulates the intracellular response of retinal rod cells to light by specifically binding to the phosphorylated light-activated form of the photoreceptor rhodopsin (P-Rh*). In order to characterize the molecular interaction between rhodopsin and arrestin, we have studied the ability of synthetic peptides from the proposed cytoplasmic loops of rhodopsin to inhibit arrestin binding. A third cytoplasmic loop peptide competed most effectively for arrestin binding to P-Rh*, exhibiting an IC50 of 34 microM, while a first cytoplasmic loop peptide weakly inhibited binding with an IC50 of approximately 1100 microM. The first and third cytoplasmic loop peptides also inhibited P-Rh* interaction with both ARR[delta (2-16)-404], an arrestin mutant that lacks residues 2-16, and ARR[1-191], a mutant that contains only the amino half of arrestin. However, the third loop peptide had an approximately 5-fold lower affinity at inhibiting the binding of ARR[1-191] to P-Rh*. While the first and third loop peptides also inhibited arrestin binding to light-activated rhodopsin and a truncated rhodopsin lacking its C-terminal sites of phosphorylation, the peptides modestly enhanced arrestin binding to phosphorylated dark rhodopsin. These results suggest that the third and, to a lesser extent, the first cytoplasmic loops of rhodopsin may play an important role in arrestin binding to light-activated forms of rhodopsin.