Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Kang, Dong Soo; Kern, Ronald C.; Puthenveedu, Manojkumar A.; Zastrow, Mark von; Williams, John C.; Benovic, Jeffrey L.

doi:10.1074/jbc.m109.023366

Cited by 116 publications

(128 citation statements)

References 58 publications

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“…2A). Each protein contains a single type II motif (42) and a recently described type III motif (43). In addition, connecdenn 1 and 2 contain two DLL motifs, whereas connecdenn 3 contains a single DLL (supplemental Fig.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…The ␤arrs can interact with downstream effectors in different modes. For example, ␤arr1 can bind between blades 1 and 2 of the clathrin ␤-propeller via an intrinsically disordered clathrin-binding box, but can also interact with a binding pocket formed by blades 4 and 5 of clathrin via an 8-amino acid splice loop found only in the long ␤arr1 isoform (99). Further insights into the allosteric regulation of ␤arr signaling have recently been provided by an NMR study that used 19 F probes in ␤arr1 to probe changes in its structure induced by different phosphopeptides derived from the V2R C terminus (100).…”

Section: Signal Transduction To Effectorsmentioning

confidence: 99%

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors

Smith¹,

Rajagopal

2016

Journal of Biological Chemistry

267

222

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The ␤-arrestins (␤arrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, ␤arrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that ␤arrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for ␤arrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. ␤-Arrestins (␤arrs)2 are ubiquitously expressed proteins that were first described for their role in desensitizing G proteincoupled receptors (GPCRs) (1). We now appreciate that these proteins are multifunctional adapter proteins that regulate a vast array of cellular functions. ␤arrs were identified through their sequence homology to visual arrestin (arrestin-1), so named because of its ability to "arrest" rhodopsin signaling in the retina (2). There are two ␤arr isoforms, ␤-arrestin1 and ␤-arrestin2 (also denoted as arrestin-2 and arrestin-3, respectively). Both are expressed ubiquitously and share 78% sequence homology (3). ␤arrs are highly conserved across species, with ϳ50% sequence homology between vertebrates and invertebrates. The other arrestins are expressed in the eye: arrestin-1 (visual arrestin) and arrestin-4 (cone arrestin) (4). There are other proteins, termed ␣-arrestins or arrestin domain-containing proteins, that share the arrestin structural fold and are involved in receptor endocytosis, although the full breadth of their functions is still emerging (5). Similar to arrestin's function in the visual system, ␤arrs were first identified for their capacity to desensitize ␤2 adrenergic receptor (␤2AR) G protein signaling following agonist stimulation (1). Through a number of investigations, it became apparent that the two ␤arr isoforms shared the capability to interact with activated GPCRs, but that they differed in terms of their expression patterns, their specificity for different GPCRs, and their functional effects (6). We now appreciate that the ␤arrs regulate a diverse array of cellular processes including MAPK signaling, receptor transactivation, receptor trafficking, and transcriptional regulation in addition to the canonical roles of GPCR desensitization and internalization (7,8). These studies have revealed the current spectrum of ␤arr-mediated cell processes downstream of GPCRs (Fig. 1). Distinct and Overlapping Roles for the ␤arrs␤arr1 and ␤arr2 knockouts are phenotypically normal and produce viable progeny, but these mice display abnormal responses to physiologic stresses (9, 10). This suggests a compensatory ability for each isoform. Nevertheless, important differences between ␤arr isoforms are present (11). Although both accumulate in the cytoplasm following overexpression, ␤arr1, but not...

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“…These initial studies focused on the β 2 -adrenergic receptor (β 2 AR) while more recent studies have demonstrated that β-arrestins promote the trafficking of many GPCRs as well as additional classes of receptors (Moore et al 2007; Shenoy and Lefkowitz 2011). Mechanistic insight into this process has revealed an essential role for the coordinated interaction of β-arrestins with the GPCR (Vishnivetskiy et al 1999, 2011), clathrin (Krupnick et al 1997; Kang et al 2009), adaptor protein 2 (AP2) (Laporte et al 1999, 2000; Kim and Benovic 2002; Schmid et al 2006; Burtey et al 2007) and phosphoinositides (Gaidarov et al 1999; Milano et al 2006). Moreover, β-arrestin binding to the GPCR appears to induce a conformational change that promotes interaction with the endocytic machinery, thereby linking the binding and trafficking events (Kim and Benovic 2002; Xiao et al 2004; Nobles et al 2007).…”

Section: β-Arrestins and Gpcr Traffickingmentioning

confidence: 99%

β-Arrestins and G Protein-Coupled Receptor Trafficking

Tian

Kang

Benovic

2013

Handbook of Experimental Pharmacology

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120

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Non-visual arrestins (β-arrestin-1 and β-arrestin-2) are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. β-arrestins are ubiquitously expressed and function to inhibit GPCR/G protein coupling, a process called desensitization, and promote GPCR trafficking and arrestin-mediated signaling. β-arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of β-arrestins with clathrin, adaptor protein 2 (AP2) and phosphoinositides. These interactions are facilitated by a conformational change in β-arrestin that is thought to occur upon binding to a phosphorylated activated GPCR. In this review, we provide an overview of the key interactions involved in β-arrestin-mediated trafficking of GPCRs.

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Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Cited by 116 publications

References 58 publications

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

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

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors

β-Arrestins and G Protein-Coupled Receptor Trafficking

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