Alison W. Gagnon

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.

Visualization of Agonist-induced Sequestration and Down-regulation of a Green Fluorescent Protein-tagged β2-Adrenergic Receptor

To date, the visualization of β2-adrenergic receptor (β2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a β2AR conjugated with green fluorescent protein (β2AR-GFP) provides the opportunity for a more extensive optical analysis of β2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of β2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of β2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of β2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different β-agonists revealed time courses of β2AR-GFP redistribution reflective of the intrinsic activity of each agonist.

Role of Arrestins in Endocytosis and Signaling of α2-Adrenergic Receptor Subtypes

We investigated the role of arrestins in the trafficking of human α2-adrenergic receptors (α2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. α2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with α2c > α2b > α2a. However, upon coexpression of arrestin-2 (β-arrestin-1) or arrestin-3 (β-arrestin-2), internalization of the α2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the α2c AR was selectively promoted by coexpression of arrestin-3, while α2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any α2-AR subtype, either in the presence or absence of arrestins. Internalization of the α2b and α2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, α2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and β2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the α2b, α2c, and to a lesser extent α2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for α2-adrenergic, epidermal growth factor, lysophosphatidic acid, or β2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.

Molecular Characterization of a Novel Human Endothelin Receptor Splice Variant

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

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.