COPI coatomer subunit α-COP interacts with the RNA binding protein Nucleolin via a C-terminal dilysine motif.

The COPI coatomer subunit α-COP has been shown to co-precipitate mRNA in multiple settings, but it was unclear whether the interaction with mRNA was direct or mediated by interaction with an adapter protein. The COPI complex often interacts with proteins via C-terminal dilysine domains. A search for candidate RNA binding proteins with C-terminal dilysine motifs yielded Nucleolin, which terminates in a KKxKxx sequence. This protein was an especially intriguing candidate as it has been identified as an interacting partner for Survival Motor Neuron protein (SMN). Loss of SMN causes the neurodegenerative disease Spinal Muscular Atrophy. We have previously shown that SMN and α-COP interact and co-migrate in axons, and that overexpression of α-COP reduced phenotypic severity in cell culture and animal models of SMA. We show here that in an mRNA independent manner, endogenous Nucleolin co-precipitates endogenous α-COP and ε-COP but not ß-COP which may reflect an interaction with the so-called B-subcomplex rather a complete COPI heptamer. The ability of Nucleolin to bind to α-COP requires the presence of the C-terminal KKxKxx domain of Nucleolin. Furthermore, we have generated a point mutant in the WD40 domain of α-COP which eliminates its ability to co-precipitate Nucleolin but does not interfere with precipitation of partners mediated by non-KKxKxx motifs such as the kainate receptor subunit 2. We propose that via interaction between the C-terminal dilysine motif of Nucleolin and the WD40 domain of α-COP, Nucleolin acts an adaptor to allow α-COP to interact with a population of mRNA.

Effects of Caffeine, a DNA Damage Response Inhibitor, on Papillomavirus Genome Replication.

Epidemiological studies have revealed that caffeinated coffee imparts a reduced risk of oropharyngeal cancer, of which human papillomavirus (HPV) is one of the causative agents. Caffeine is a known inhibitor of the DNA damage response (DDR) pathway. We sought to test the effects of caffeine on the early replication of the HPV31 virus. It has been reported that the inhibition of several factors necessary for the DDR during the differentiation-dependent stage of HPV block genome amplification, while the HPV genome maintenance replication was unaffected. We first studied the effects of caffeine in the earliest stages of viral infection. Using pseudo-virions (PsV) expressing an m-Cherry reporter gene and quasi-virions (QsV) containing HPV31 genomes to mediate the infection, we found no evidence that caffeine impeded the viral entry; however, the infected cells displayed a reduced HPV copy number. In contrast, caffeine exposure increased the copy number of HPV31 episomes in the transient transfection assays and in the CIN612E cells that stably maintain viral episomes. There was a concomitant increase in the steady state levels of the HPV31 E1 and E2 transcripts, along with increased E2 loading at the viral origin of replication (ori). These results suggest that the caffeine-mediated inhibition of the DDR reduces viral genome replication in the early stage of infection, in contrast to the maintenance stage, in which the inhibition of the DDR may lead to an increase in viral amplicon replication.

Regulation of the Human Papillomavirus Lifecyle through Post-Translational Modifications of the Viral E2 Protein.

The human papillomavirus (HPV) is a DNA tumor virus that infects cutaneous and mucosal epithelia where high-risk (HR) HPV infections lead to cervical, oropharyngeal, and anogenital cancers. Worldwide, nearly 5% of all cancers are caused by HR HPV. The viral E2 protein is essential for episomal replication throughout the viral lifecycle. The E2 protein is regulated by phosphorylation, acetylation, sumoylation, and ubiquitination. In this mini-review, we summarize the recent advancements made to identify post translational modifications within E2 and their ability to control viral replication.

Optimization of human papillomavirus-based pseudovirus techniques for efficient gene transfer.

Human papillomavirus (HPV) L1 and L2 capsid proteins self-assemble into virions capable of efficiently packaging either its 8 kilobase genome or non-viral DNA. The ability of HPV capsids to package non-viral DNA makes these a useful tool for delivering plasmids to study proteins of interest in a variety of cell types. We describe optimization of current methods and present new protocols for using HPV capsids to deliver non-viral DNA thereby providing an alternative to DNA transfection. Using keratinocyte generated extracellular matrices can enhance infection efficiency in keratinocytes, hepatocytes and neuronal cells. Furthermore, we describe a suspension-based efficient technique for infecting different cell types.

Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance.

Several serine and threonine residues of the papillomavirus early E2 protein have been found to be phosphorylated. In contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one in HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this study, we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the nonphosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither binds the C terminus of Brd4, but in all other aspects the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in vitro assay, while HPV-31 Y102E binds E1 and was able to replicate, albeit at lower levels than wild type. To examine the effect of E2 mutations under more native-like infection conditions, a neomycin-selectable marker was inserted into L1/L2 of the HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days posttransfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines.IMPORTANCE Posttranslational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

Papillomavirus E2 protein is regulated by specific fibroblast growth factor receptors.

The papillomavirus (PV) E2 protein activates transcription and replication by recruiting cellular proteins and the E1 DNA helicase to their binding sites in the viral genome. We recently demonstrated that phosphorylation of tyrosine 102 in the bovine papillomavirus (BPV-1) E2 protein restricts these activities and that fibroblast growth factor receptor-3 (FGFR3) tyrosine kinase binds PV E2. Expression of FGFR3 decreased viral replication with both wild-type and the phenylalanine substitution at position 102, inferring that another kinase targets Y102. Here we tested FGFR- 1, -2 and -4 for association with PV E2 proteins. FGFR2 but not FGFR1 or FGFR4 co-immunoprecipitated with BPV-1 E2. We found that FGFR2 suppressed replication but did not depend on phosphorylation of BPV-1 Y102. HPV-16 and -31 E2 interacted with FGFR1, -2, and -4. These results imply that the expression and activity of FGF receptors in epithelial cells can regulate the function of E2 in viral replication.

Kinase Activity of Fibroblast Growth Factor Receptor 3 Regulates Activity of the Papillomavirus E2 Protein.

The papillomavirus (PV) E2 protein is a DNA binding, protein interaction platform that recruits viral and host factors necessary for transcription and replication. We recently discovered phosphorylation of a tyrosine (Y102) in bovine PV (BPV) E2. To identify the responsible factor, we tested several candidate tyrosine kinases that are highly expressed in keratinocytes for binding to BPV-1 E2. Fibroblast growth factor receptor 3 (FGFR3) coimmunoprecipitated with the BPV-1 E2 protein, as did human papillomavirus 31 (HPV-31) E2, which also colocalized with FGFR3 within the nucleus. A constitutively active mutant form of FGFR3 decreased BPV-1 and HPV-31 transient replication although this result also occurred in a BPV-1 E2 mutant lacking a previously identified phosphorylation site of interest (Y102). Furthermore, FGFR3 depletion in cell lines that maintain HPV-31 episomes increased viral copy number. These results suggest that FGFR3 kinase activity may regulate the PV reproductive program through phosphorylation of the E2 protein although this is unlikely to occur through the Y102 residue of HPV E2.IMPORTANCE The papillomavirus (PV) is a double-stranded DNA tumor virus infecting cervix, mouth, and throat tissues. The viral protein E2 is responsible for the replication of the virus. Understanding the mechanisms of the replicative life cycle of the virus may bring to light direct targets and treatments against viral infection. We recently found that the fibroblast growth factor receptor 3 (FGFR3) interacts with and mediates PV E2 function through phosphorylation of the E2 protein. Our study suggests that the function of the E2 protein may be regulated through a direct FGFR3 target during the maintenance stage of the PV life cycle.

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions.

Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-to-glutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection. IMPORTANCE: Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

Altered mRNA Splicing in SMN-Depleted Motor Neuron-Like Cells.

Spinal muscular atrophy (SMA) is an intractable neurodegenerative disease afflicting 1 in 6-10,000 live births. One of the key functions of the SMN protein is regulation of spliceosome assembly. Reduced levels of the SMN protein that are observed in SMA have been shown to result in aberrant mRNA splicing. SMN-dependent mis-spliced transcripts in motor neurons may cause stresses that are particularly harmful and may serve as potential targets for the treatment of motor neuron disease or as biomarkers in the SMA patient population. We performed deep RNA sequencing using motor neuron-like NSC-34 cells to screen for SMN-dependent mRNA processing changes that occur following acute depletion of SMN. We identified SMN-dependent splicing changes, including an intron retention event that results in the production of a truncated Rit1 transcript. This intron-retained transcript is stable and is mis-spliced in spinal cord from symptomatic SMA mice. Constitutively active Rit1 ameliorated the neurite outgrowth defect in SMN depleted NSC-34 cells, while expression of the truncated protein product of the mis-spliced Rit1 transcript inhibited neurite extension. These results reveal new insights into the biological consequence of SMN-dependent splicing in motor neuron-like cells.

Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses.

UNLABELLED: E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif. IMPORTANCE: Two early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some viruses use Cul2 to form the complex, while others use Cul5. In the present study, we expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses encode proteins containing the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of the Human mastadenovirus A, F, and G species. We also identified previously unrecognized E4orf6 genes in the aviadenoviruses that encode proteins containing motifs permitting formation of the ubiquitin ligase.

α-COP binding to the survival motor neuron protein SMN is required for neuronal process outgrowth.

Spinal muscular atrophy (SMA), a heritable neurodegenerative disease, results from insufficient levels of the survival motor neuron (SMN) protein. α-COP binds to SMN, linking the COPI vesicular transport pathway to SMA. Reduced levels of α-COP restricted development of neuronal processes in NSC-34 cells and primary cortical neurons. Remarkably, heterologous expression of human α-COP restored normal neurite length and morphology in SMN-depleted NSC-34 cells in vitro and zebrafish motor neurons in vivo. We identified single amino acid mutants of α-COP that selectively abrogate SMN binding, retain COPI-mediated Golgi-ER trafficking functionality, but were unable to support neurite outgrowth in cellular and zebrafish models of SMA. Taken together, these demonstrate the functional role of COPI association with the SMN protein in neuronal development.

Analysis of the Cullin binding sites of the E4orf6 proteins of human adenovirus E3 ubiquitin ligases.

UNLABELLED: E4orf6 proteins of human adenoviruses form Cullin-based E3 ubiquitin ligase complexes that degrade cellular proteins, which impedes efficient viral replication. These complexes also include the viral E1B55K product, which is believed to recruit most substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as serotypes representing some species form Cul5-based complexes (species B2, C, D, and E), whereas others utilize Cul2 (species A and F). Adenovirus type 16 (Ad16; species B1) binds significant levels of both. In this report, we show that the Cul2 binding sequence in E4orf6 of Ad12 (species A) and Ad40 (species F) resembles the cellular consensus Cul2 box. Mutation within this Cul2 box prevents binding not only of Cul2 but also in some cases Elongin C and reduces the ability to degrade target proteins, such as Mre11 and p53. A comparable Cul2 box is not present in E4orf6 of Ad5 and other serotypes that bind Cul5; however, creation of this Cul2 box sequence in Ad5 E4orf6 promoted binding to Cul2 and Cul2-dependent degradation of Mre11. E4orf6 of Ad16 also binds Cul2; however, unlike Ad40, it does not contain an Ad12-like Cul2 box, suggesting that Ad16 binds Cul2 in a unique but perhaps nonfunctional manner, as only Cul5 binding complexes appeared able to degrade Mre11. Our extensive analyses have thus far failed to identify a consensus Cul5 binding sequence, suggesting that association occurs via a novel and perhaps complex pattern of protein-protein interactions. Nevertheless, the identification of the Cul2 box may allow prediction of Cullin specificity for all E4orf6-containing Adenoviridae. IMPORTANCE: The work described in this paper is a continuation of our in-depth studies on the Cullin-based E3 ligase complexes formed by the viral E4orf6 and E1B55K proteins of all human adenoviruses. This complex induces the degradation of a growing series of cellular proteins that impede efficient viral replication. Some human adenovirus species utilize Cul5, whereas others bind Cul2. In this paper, we are the first to identify the E4orf6 Cul2 binding site, which conforms in sequence to a classic cellular Cul2 box. Ours is the first detailed biochemical and genetic analysis of a Cul2-based adenovirus ligase and provides insights into both the cooperative interactions in forming Cullin-based ligases as well as the universality of formation of all adenovirus ligase complexes. Our work now permits future analysis of the evolutionary significance of the ligase complex, work that is currently in progress in our lab.

Role of E1B55K in E4orf6/E1B55K E3 ligase complexes formed by different human adenovirus serotypes.

The E4orf6 protein of serotypes representing all human adenovirus species forms Cullin-based E3 ubiquitin ligase complexes that facilitate virus infection by inducing degradation of cellular proteins that impede efficient viral replication. This complex also includes the viral E1B55K product believed to bind and introduce substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as some serotypes form Cul5-based complexes whereas others utilize Cul2. Significant variations in substrate specificities also exist among serotypes, as some degrade certain substrates very efficiently whereas others induce more modest or little degradation. As E1B55K is believed to function as the substrate acquisition component of the ligase, we undertook studies to compare the ability of representative E1B55K proteins to bind substrates with the efficacy of degradation by their respective E4orf6-based ligases. Interestingly, although efficient degradation in some cases corresponded to the ability of E1B55K to bind to or relocalize substrates, there were several examples of substrates that bound efficiently to E1B55K but were not degraded and others in which substrates were degraded even though binding to E1B55K was low or undetectable. These results suggest that transient interactions with E1B55K may be sufficient for efficient substrate degradation and that binding alone is not sufficient, implying that the orientation of the substrate in the ligase complex is probably crucial. Nevertheless, we found that the substrate specificity of certain E4orf6-based ligases could be altered through the formation of hybrid complexes containing E1B55K from another serotype, thus confirming identification of E1B55K as the substrate acquisition component of the complex.

The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

The E4orf6/E1B55K E3 ubiquitin ligase complexes of human adenoviruses exhibit heterogeneity in composition and substrate specificity.

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin α3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin α3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.

E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection.

The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC complex. These observations suggest that dissociation of the ligase IV/XRCC4 complex occurs at an early stage in E4 34k-mediated degradation of ligase IV and indicate a role for E4 34k in dissociation of the ligase IV/XRCCC4 complex. Expression of E4 34k alone was not sufficient to dissociate the ligase IV/XRCC4 complex, which indicates a requirement for an additional, as yet unidentified, factor in E1B 55k-independent dissociation of the ligase IV/XRCC4 complex.

Comparison of the relative efficacy and potency of mu-opioid agonists to activate Galpha(i/o) proteins containing a pertussis toxin-insensitive mutation.

Pertussis toxin (PTX)-insensitive mutants of Galpha(i/o) proteins expressed in C6mu cells were used to examine the hypothesis that there are agonist-specific conformational states of the mu-opioid receptor with coupling preferences to different Galpha(i/o) subtypes, as measured by the degree of stimulation of [(35)S]guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) binding. Binding of [(35)S]GTPgammaS to endogenous Galpha(i/o) proteins stimulated by the full mu-opioid agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was completely blocked by overnight treatment with 100 ng/ml PTX. Treatment for 4 h with lower concentrations led to a PTX-dependent reduction in the maximal effect of DAMGO but no alteration in the potency of DAMGO or morphine nor in the relative maximal effect (relative efficacy) of the partial agonists morphine and buprenorphine compared with the full agonist DAMGO. Using PTX-insensitive Galpha mutants in which the PTX-sensitive cysteine was replaced with isoleucine, the potency for a series of mu-opioid agonists was highest in cells expressing Galpha(i3) and Galpha(o) and lowest with Galpha(i1) and Galpha(i2), with no significant change in the order of potency, namely, etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine. The order of agonist relative efficacy, etorphine = DAMGO = endomorphin-1 = endomorphin-2 = fentanyl > or = morphine > or = meperidine > buprenorphine > or = nalbuphine, was also the same across all of the PTX-insensitive Galpha(i/o) subtypes. Highest relative efficacy to stimulate [(35)S]GTPgammaS binding was seen with Galpha(i3). Consequently, reported observations of agonist-directed trafficking at mu-opioid receptors most likely involve non-PTX-sensitive Galpha protein mechanisms.