Arrestin/Clathrin Interaction

Abstract

Receptor-mediated endocytosis is a pathway by which plasma membrane receptors are internalized selectively into cells through clathrin-coated pits (for review, see Refs. 1 and 2). Receptors are either internalized constitutively (e.g. some nutrient receptors) or internalized preferentially following ligand binding (e.g. some hormone receptors). Among those that belong to the latter category are several G protein-coupled receptors (GPRs).1 Many GPRs are phosphorylated rapidly upon agonist activation, and it is this phosphorylation event that leads to the selective binding of a family of proteins termed arrestins. To date there are four known arrestins: two visual arrestins (arrestin and cone arrestin) and two nonvisual arrestins (␤-arrestin and arrestin3; for review, see Ref.3). One role of arrestin binding is to uncouple the GPR from its cognate heterotrimeric G protein (desensitization), probably through direct competition with the G protein for the activated GPR (4). Following desensitization, the receptor is believed to be translocated into an intracellular compartment (5), a process commonly referred to as sequestration. Interestingly, sequestration also appears to be required for resensitization of receptors by allowing for receptor dephosphorylation and recycling (6, 7).Studies of the m2 muscarinic acetylcholine receptor (8) and a sequestration-deficient mutant form of the ␤ 2 -adrenergic receptor (9) have implicated GPR kinases and arrestins in GPR sequestration. Recent work from our laboratories has provided a plausible molecular mechanism for this internalization process, in which arrestins function as clathrin adaptors in the uptake of ␤ 2 -adrenergic (10) and other 2 GPRs. We found that nonvisual arrestins bind to clathrin with a K d of 10 -60 nM, comparable to that of the clathrin associated protein AP-2 (12, 13). Moreover, ␤ 2 -adrenergic receptor, ␤-arrestin, and clathrin colocalize in vivo upon agonist addition in an arrestin-dependent manner, indicating that the arrestin/clathrin interaction observed in vitro likely occurs in vivo in the presence of an activated receptor. These results suggest that the arrestin/ clathrin interaction is of central importance in regulating GPR trafficking.Clathrin, the major structural component of the coated pit lattice, consists of three heavy chains (M r Ϸ 192,000) and three light chains (M r Ϸ 26,000) (for review, see Ref. 14). By limited proteolysis of preformed clathrin cages, the heavy chain has been shown to comprise two distinct domains (15). An Ϸ50-kDa amino-terminal globular region, the terminal domain (TD), lies at the distal end of each triskelion leg. The remaining Ϸ140 kDa of the heavy chain forms the triskelion core, consisting of the vertex, proximal leg, and a portion of the distal leg of the clathrin trimer. One clathrin light chain is associated with each proximal leg. Based on electron microscopic studies and models of the polygonal lattice, a clathrin hub lies at each vertex, and each lattice edge is formed by the superposition of two distal a...

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

“…It is interesting to note, however, that previous work that measured arrestin binding to clathrin mutants that were disrupted in the LX(D/E) binding pocket (e.g. F91A, K96E, and K98E) reported a complete disruption of arrestin3 binding but only a 65-80% loss of arrestin2L binding (24). Thus, although not noted in the paper, these previous results also suggest that a second clathrin binding domain is present in arrestin2L.…”

“…Each construct was purified as previously described with minor modifications (10,24). Selenomethionyl-labeled clathrin-(1-363) was prepared and purified similar to that previously described (25,27) except the cells were grown in a selenomethionine minimum media (28).…”

“…Importantly, the mutation of this motif in arrestin3 and its deletion in arrestin2 significantly disrupts clathrin binding and receptor endocytosis (14,19). A mutagenesis study of clathrin localized an arrestin binding site to the N-terminal domain of the clathrin heavy chain, specifically residues Glu-89, Lys-96, and Lys-98 (24). Moreover, a crystal structure of clathrin-(1-363) in complex with an arrestin3 peptide (residues 369 -381) supports the mutagenesis data and the predicted location of the arrestin-clathrin interaction site (25).…”

Interaction of Two Structurally Distinct Sequence Types with the Clathrin Terminal Domain β-Propeller

“…Here, we show that the two dissimilar clathrin-binding sequences present in amphiphysin and in epsin (15) can function together in vitro. We also show that, unlike arrestin with only a single type I sequence (48), amphiphysin can generate pelletable clathrin assemblies within 30 min, even at neutral pH. AP180 and epsin both have epsin N-terminal homology (ENTH) domains at the amino terminus (49), and each binds to phosphoinositides directly (10,50).…”

Identification of a Motif in the Carboxyl Terminus of CXCR2 That Is Involved in Adaptin 2 Binding and Receptor Internalization

“…In a word, for many GPCRs, receptor phosphorylation has been suggested to be required for the receptor internalization, and β-arrestins have been proposed to play an important role in this process. Although β-arrestins have been shown to bind to clathrin with high affinity (15), they are not constitutively associated with clathrin-coated vesicles (16), nor do they promote clathrin-coat assembly (17). These studies imply that some other adaptor proteins are also involved in this process.…”