Jason G. Krupnick

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.

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.

Mechanism of quenching of phototransduction. Binding competition between arrestin and transducin for phosphorhodopsin.

Quenching of phototransduction in retinal rod cells involves phosphorylation of photoactivated rhodopsin by the enzyme rhodopsin kinase followed by binding of the protein arrestin. Although it has been proposed that the mechanism of arrestin quenching of visual transduction is via steric exclusion of transducin binding to phosphorylated light-activated rhodopsin (P-Rh*), direct evidence for this mechanism is lacking. In this study, we investigated both the role of rhodopsin phosphorylation in modulating its interaction with arrestin and transducin and the proposed binding competition between arrestin and transducin for P-Rh*. While the β-adrenergic receptor kinase promotes significant arrestin binding to rhodopsin at a phosphorylation stoichiometry of ≥2 mol/mol, rhodopsin kinase promotes arrestin binding at a stoichiometry of ∼0.9 mol/mol. Moreover, while β-adrenergic receptor kinase phosphorylation of rhodopsin only modestly decreases transducin binding and activation, rhodopsin kinase phosphorylation of rhodopsin significantly decreases transducin binding and activation. Finally, arrestin competes effectively with transducin for binding to P-Rh* (50% inhibition at ∼1:1 molar ratio of arrestin:transducin) but has no effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh*), paralleling the functional inhibition by arrestin on P-Rh*-stimulated transducin activation (50% inhibition at ∼1.7:1 molar ratio of arrestin:transducin). These results demonstrate that a major role of rhodopsin phosphorylation is to promote high-affinity arrestin binding and decrease transducin binding thus allowing arrestin to effectively compete with transducin for binding to photoactivated rhodopsin.

Signaling and Phosphorylation-impaired Mutants of the Rat Follitropin Receptor Reveal an Activation- and Phosphorylation-independent but Arrestin-dependent Pathway for Internalization

We have previously shown that the rat follitropin receptor (rFSHR) expressed in transfected cells becomes phosphorylated upon stimulation of the cells with agonist or a phorbol ester. Peptide mapping and mutagenesis studies have also shown that the agonist- or phorbol ester-induced phosphorylation of the rFSHR maps to Ser/Thr residues present in the first and third intracellular loops. The experiments presented herein were initially designed to test for the presence of additional phosphorylation sites on the second intracellular loop of the rFSHR. Analysis of two new mutants in which the two threonines in the second intracellular loop (rFSHR-2L) or the two threonines in the second intracellular loop and the seven Ser/Thr residues in the third intracellular loop (rFSHR-2L + 3L) were mutated showed that one or more of the two threonines in the second intracellular loop are phosphorylated in response to phorbol ester, but not in response to agonist stimulation. Since rFSHR-2L and rFSHR-2L + 3L displayed a reduction in agonist-induced signaling, two additional mutants (rFSHR-D389N and rFSHR-Y530F) were constructed in an attempt to better understand the relationship between the agonist-induced activation, phosphorylation, and internalization of the rFSHR. These point mutations impaired agonist-stimulated signal transduction and abolished agonist-induced phosphorylation. Co-transfection studies revealed that the phosphorylation of these mutants can be rescued by overexpression of G protein-coupled receptor kinase 2, but this increased phosphorylation only rescues the internalization of rFSHR-D389N. The internalization of both mutants could be rescued by overexpression of arrestin-3, however. Taken together, these results argue that the agonist-induced activation and phosphorylation of the rFSHR are not essential for internalization. while the interaction of the rFSHR with a nonvisual arrestin is essential for internalization.

Mutation of Individual Serine Residues in the C-terminal Tail of the Lutropin/Choriogonadotropin Receptor Reveal Distinct Structural Requirements for Agonist-induced Uncoupling and Agonist-induced Internalization

We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20–40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.

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.