Jeffrey L. Benovic

The E3 Ubiquitin Ligase AIP4 Mediates Ubiquitination and Sorting of the G Protein-Coupled Receptor CXCR4

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.

Trafficking of the HIV Coreceptor CXCR4 ROLE OF ARRESTINS AND IDENTIFICATION OF RESIDUES IN THE C-TERMINAL TAIL THAT MEDIATE RECEPTOR INTERNALIZATION

The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1α). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1α and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA- and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1α. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA- and SDF-1α-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

G-protein-coupled receptors: turn-ons and turn-offs

Advances in the study of G-protein-coupled receptor regulation have provided novel insights into the role of G-protein-coupled receptor kinases and arrestins in this process. Of particular interest are recent studies that have dramatically expanded the known cellular functions of these molecules to include roles in receptor endocytosis and activation of MAP kinase signalling pathways.

Regulation of G Protein-Coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) specifically interact with the agonist-activated form of G protein-coupled receptors (GPCRs) to effect receptor phosphorylation and desensitization. Recent studies demonstrate that GRK function is a highly regulated process, and it is perhaps in this manner that a handful of GRKs (7 have been identified to date) are able to regulate the responsiveness of numerous GPCRs in a given cell type in a coordinated manner. The mechanisms by which GRK activity is regulated can be divided into 3 categories: 1) subcellular localization; 2) alterations in intrinsic kinase activity; and 3) alterations in GRK expression levels. This review will summarize our current understanding of each of these regulatory processes, and offer explanations as to how such mechanisms influence GPCR regulation under various physiologic conditions.

The ins and outs of G protein-coupled receptor trafficking

Abstract All cells are faced with the complexity of converting a myriad of extracellular stimuli to intracellular signals and appropriate biological responses. This process involves a series of highly orchestrated molecular events, many of which occur at the plasma-membrane interface. Endocytosis is one such event, and functions to transport numerous small molecules and protein cargo. For G protein-coupled receptors (GPCRs), endocytosis serves as a mechanism to regulate cell-surface-receptor levels, thereby, contributing to the regulation of hormonal responsiveness. The trafficking of GPCRs through the endocytic pathway involves three main steps: recruitment of receptors to discrete endocytic sites, internalization and intracellular sorting. Many recent studies have advanced our understanding of these steps. Novel mechanistic insight into the recruitment of GPCRs to sites of endocytosis has been gained and the discovery of novel protein–protein interactions, which define more clearly the processes of internalization and endocytic sorting, have been characterized. In addition, a role for ubiquitin in internalization and sorting to the degradative pathway has emerged.

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 β2-adrenergic receptor (β2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of β-arrestin (β-arrestin (319–418)) to function as a dominant-negative with respect to β2AR internalization and compared its properties with those of β-arrestin and β-arrestin-V53D, a previously reported dominant-negative mutant.In vitro studies demonstrated that β-arrestin-V53D bound better to clathrin than β-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas β-arrestin (319–418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the β2AR in HEK293 cells, β-arrestin (319–418) effectively inhibited agonist-promoted receptor internalization, whereas β-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of β2AR internalization by β-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both β-arrestin (319–418) and β-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of β-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.

G-protein-coupled receptor kinases

Rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) catalyse the phosphorylation of the activated forms of the G-protein-coupled receptors, rhodopsin and the beta 2-adrenergic receptor (beta 2AR), respectively. The interaction between receptor and kinase is independent of second messengers and appears to involve a multipoint attachment of kinase and substrate with the specificity being restricted by both the primary amino acid sequence and conformation of the substrate. Kinetic, functional and sequence information reveals that rhodopsin kinase and beta ARK are closely related, suggesting they may be members of a family of G-protein-coupled receptor kinases.

Internalization of the TXA2 Receptor α and β Isoforms ROLE OF THE DIFFERENTIALLY SPLICED COOH TERMINUS IN AGONIST-PROMOTED RECEPTOR INTERNALIZATION

Thromboxane A2(TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, α (343 residues) and β (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rβ, but not the TXA2Rα, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rβ is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the α and β isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rβ did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rβ is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.

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 arrestins function as an adaptor. High‐affinity binding sites for phosphoinositides in β‐arrestin (arrestin2) and arrestin3 (β‐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 β2‐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.

Safe use of the CXCR4 inhibitor ALX40-4C in humans

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.