James H. Keen

Spatial control of coated-pit dynamics in living cells

Here we visualize new aspects of the dynamics of endocytotic clathrin-coated pits and vesicles in mammalian cells by using a fusion protein consisting of green fluorescent protein and clathrin light chain a. Clathrin-coated pits invaginating from the plasma membrane show definite, but highly limited, mobility within the membrane that is relaxed upon treatment with latrunculin B, an inhibitor of actin assembly, indicating that an actin-based framework may be involved in the mobility of these pits. Transient, motile coated vesicles that originate from coated pits can be detected, with multiple vesicles occasionally appearing to emanate from a single pit. Despite their seemingly random distribution, coated pits tend to form repeatedly at defined sites while excluding other regions. This spatial regulation of coated-pit assembly and function is attributable to the attachment of the coated pits to the membrane skeleton.

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.

The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1Rs properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

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.

The Class II Phosphoinositide 3-Kinase C2α Is Activated by Clathrin and Regulates Clathrin-Mediated Membrane Trafficking

Phosphoinositides play key regulatory roles in vesicular transport pathways in eukaryotic cells. Clathrin-mediated membrane trafficking has been shown to require phosphoinositides, but little is known about the enzyme(s) responsible for their formation. Here we report that clathrin functions as an adaptor for the class II PI 3-kinase C2alpha (PI3K-C2alpha), binding to its N-terminal region and stimulating its catalytic activity, especially toward phosphorylated inositide substrates. Further, we show that endogenous PI3K-C2alpha is localized in coated pits and that exogenous expression affects clathrin-mediated endocytosis and sorting in the trans-Golgi network. These findings provide a mechanistic basis for localized inositide generation at sites of clathrin-coated bud formation, which, with recruitment of inositide binding proteins and subsequent synaptojanin-mediated phosphoinositide hydrolysis, may regulate coated vesicle formation and uncoating.

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.

A Functional Phosphatidylinositol 3,4,5-Trisphosphate/Phosphoinositide Binding Domain in the Clathrin Adaptor AP-2 α Subunit. IMPLICATIONS FOR THE ENDOCYTIC PATHWAY

Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4,5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble D-myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 α subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 α sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian α, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4,5-trisphosphate to AP-2 inhibits the proteins clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors.

Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.

THE ALPHA CHAIN OF THE AP-2 ADAPTOR IS A CLATHRIN BINDING SUBUNIT

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated β2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated αa exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of αa bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a ≈29-kDa αa clathrin-binding fragment that remained tightly cage-associated. Translated αa also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated β2 subunit. Clathrin binding by both the α and β subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H.(1987) Cell Biol. 105, 1989). It also [Abstract] raises the possibility that the α-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other α functions.

ROLE OF ARRESTINS IN G-PROTEIN-COUPLED RECEPTOR ENDOCYTOSIS

Publisher Summary β2AR activation by catecholamines initiates a cascade of events that culminate in the cyclic adenosine monophosphate-dependent phosphorylation of multiple cell specific target proteins. Within seconds to minutes after activation by agonist, β2AR becomes phosphorylated by the β-adrenergic receptor kinase (βARK). β2AR phosphorylation by βARK promotes the binding of another protein, termed β-arrestin, to the receptor, which effectively uncouples the β2AR from the stimulatory G-protein and attenuates signaling. β2AR uncoupling is rapidly followed by a loss, or sequestration, of cell surface β2ARs into an intracellular compartment distinct from the plasma membrane. Recent studies suggest that β2AR internalization may be important for receptor resensitization, via a process that involves dephosphorylation and recycling of the receptor back to the plasma membrane. There is general agreement that GRs are physically internalized into cells in an agonist-dependent manner, and that this process may occur by both clathrin- and non-clathrin-mediated processes. For β2AR in particular, the available evidence supports receptor internalization predominantly through clathrincoated pits. Based on the studies described, it is hypothesized that arrestins, which bind directly to activated phosphorylated receptors, play a pivotal role in β2AR internalization via their interaction with some component of the clathrin-coated pit. To explore this possibility, studies examined whether arrestins interact with clathrin, the major structural protein of coated pits. In initial studies, in vitro translated radiolabeled β-arrestin and arrestin 3 are found to bind specifically to clathrin cages, while visual arrestin showed no appreciable binding. To determine whether β-arrestin and arrestin 3s interaction with clathrin occured directly, the binding of purified recombinant arrestins to clathrin in several assembled forms is assessed. The ability of β-arrestin and arrestin 3 to bind both GRs and clathrin with high affinity suggests that nonvisual arrestins likely function as adaptor proteins to promote receptor localization in clathrin-coated pits. To address this question in intact cell studies, it is assessed whether β2ARs are localized in clathrincoated pits in an agonist- and β-arrestin-dependent manner.