Src-dependent phosphorylation of β2-adaptin dissociates the β-arrestin–AP-2 complex

Fessart, Delphine; Simaan, May; Zimmerman, Brandon; Comeau, Jonathan; Hamdan, Fadi F.; Wiseman, Paul W.; Bouvier, Michel; Laporte, Stéphane A.

doi:10.1242/jcs.03444

Cited by 39 publications

(28 citation statements)

References 36 publications

(53 reference statements)

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“…Expression of a Y497F mutant of dynamin1 impairs the internalization of both the ␤ 2 adrenergic and the M 2 muscarinic receptors (Ahn et al, 1999;Werbonat 312 LUTTRELL AND GESTY-PALMER et al, 2000). The ␤2 adaptin subunit of AP-2 is also regulated by arrestin-dependent Src phosphorylation (Fessart et al, 2005(Fessart et al, , 2007Zimmerman et al, 2009). c-Src stabilizes the constitutive association of arrestin3 and ␤2 adaptin independent of its kinase activity.…”

Section: Receptor Endocytosis and Vesicle Traffickingmentioning

confidence: 99%

Beyond Desensitization: Physiological Relevance of Arrestin-Dependent Signaling

2010

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Section: Receptor Endocytosis and Vesicle Traffickingmentioning

confidence: 99%

Beyond Desensitization: Physiological Relevance of Arrestin-Dependent Signaling

2010

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“…3A). This suggests that the interaction between connecdenn 2 and AP-2 may be transient in nature, such as is the case for Eps15 (10), or alternatively that its recruitment onto CCVs may be subject to regulation, as is the case for other ␤2-ear-binding proteins, such as ␤arrestins and PIPK␥661, which in their inactive state are found in a cytoplasmic pool, only associating with AP-2 upon activation (13,20,41,45,59).…”

Section: Identification Of Key Residues In the Connecdenn 2-binding Mmentioning

confidence: 99%

The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery

Marat¹,

McPherson

2010

Journal of Biological Chemistry

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Rabs constitute the largest family of monomeric GTPases, yet for the majority of Rabs relatively little is known about their activation and recruitment to vesicle-trafficking pathways. We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as a guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35, whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2. Characterization of connecdenn 2 reveals binding to the ␤2-ear of AP-2 on a site that overlaps with that used by the autosomal recessive hypercholesterolemia protein and ␤arrestin, although the sequence used by connecdenn 2 is unique. Loss of connecdenn 2 function through small interference RNA knockdown results in an enlargement of early endosomes, similar to what is observed upon loss of Rab35 activity. Our studies reveal connecdenn DENN domains as generalized GEFs for Rab35 and identify a new AP-2-binding motif, demonstrating a complex link between the clathrin machinery and Rab35 activation. Clathrin-mediated endocytosis (CME)3 is a major mechanism for internalization of proteins and lipids. Clathrin-coated vesicles (CCVs) form at the plasma membrane and deliver their cargo to early endosomes from were it either recycles or is targeted for degradation. Central to the formation of CCVs at the plasma membrane is the clathrin adaptor protein 2 (AP-2). AP-2 is a heterotetramer consisting of two large subunits, ␣ and ␤2, a medium sized 2 subunit, and a small 2 subunit. The ␣ and ␤2 subunits each contain an N-terminal trunk domain, which along with 2 and 2 form the core of AP-2. In addition, ␣ and ␤2 contain C-terminal, ϳ30-kDa ear (or appendage) domains, separated from the trunk by flexible linkers. The core of AP-2 targets it to the plasma membrane, whereas the ␤2-ear and linker bind and promote the polymerization of clathrin. The ␣-and ␤2-ear domains serve as recruitment hubs, binding accessory proteins and bringing them to sites of CCV formation (1).Despite low sequence homology, the ␣-and ␤2-ears have a similar bi-lobed structure, consisting of N-terminal sandwich and C-terminal platform subdomains (2-4). Each of the four subdomains binds to specific peptide motifs, allowing the ears to recruit a wide variety of accessory proteins. The presence of four distinct binding sites, each of which has a different binding affinity, is likely important for the correct temporal ordering during the recruitment of endocytic accessor...

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“…Immunoprecipitation and Western Blotting-Immunoprecipitation experiments were carried out as described previously (65). Immunoprecipitated proteins were separated on 6% SDS-PAGE, whereas proteins from total cell lysates were separated on 10% SDS-PAGE before transfer onto nitrocellulose membranes.…”

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confidence: 99%

Unraveling G Protein-coupled Receptor Endocytosis Pathways Using Real-time Monitoring of Agonist-promoted Interaction between β-Arrestins and AP-2

Hamdan

Rochdi

Breton

et al. 2007

Journal of Biological Chemistry

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The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves ␤-arrestin and clathrin-coated pits. However, both ␤-arrestin-and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that ␤-arrestin binding to the ␤2 subunit of the clathrin adaptor AP-2 (␤2-adaptin) is needed for the ␤-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrinindependent pathways, agonist stimulation promoted ␤-arrestin 1 and 2 interaction with ␤2-adaptin, indicating a ␤-arrestinand clathrin-dependent endocytic process. Detailed analyses of ␤-arrestin interactions with both the receptor and ␤2-adaptin also allowed us to demonstrate that recruitment of ␤-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between ␤-arrestins and ␤2-adaptin. However, both receptors recruited ␤-arrestins upon agonist stimulation, suggesting a ␤-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based ␤-arrestin/␤2-adaptin interaction assay represents a novel biosensor to assess receptor activation. G protein-coupled receptors (GPCRs)7 are seven transmembrane domain receptors that constitute the largest family of cell surface proteins involved in signal transduction. In humans, it is estimated that GPCRs are encoded by ϳ800 distinct genes that control a variety of important physiological responses (1). Following agonist binding, GPCRs undergo conformational changes that regulate the activity of downstream effector systems to mediate various cellular responses. The extent and duration of GPCR signaling is tightly regulated by mechanisms that terminate the initial signaling and later re-establish the capacity of the receptors to respond to new agonist exposure. The removal of GPCRs from the cell surface, also known as internalization or endocytosis, plays an important role in these processes (2, 3). For most GPCRs, rapid feedback desensitization is initiated by G protein-coupled receptor kinases that phosphorylate agonist-occupied GPCRs to create high affinity binding sites for ␤-arrestins, which in turn uncouple the receptor from its cognate G protein (reviewed in Ref. 4). ␤-Arrestins also target rece...

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Src-dependent phosphorylation of β2-adaptin dissociates the β-arrestin–AP-2 complex

Cited by 39 publications

References 36 publications

Beyond Desensitization: Physiological Relevance of Arrestin-Dependent Signaling

Beyond Desensitization: Physiological Relevance of Arrestin-Dependent Signaling

The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery

Unraveling G Protein-coupled Receptor Endocytosis Pathways Using Real-time Monitoring of Agonist-promoted Interaction between β-Arrestins and AP-2

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