Transition of Arrestin into the Active Receptor-binding State Requires an Extended Interdomain Hinge

Abstract

Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptorbinding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/ rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptorbound) arrestin conformation is essentially the same in both wild type and mutant forms.The signaling by a vast family of G protein-coupled receptors is quenched by the binding of arrestin proteins to activated receptors that have been phosphorylated by specific receptor kinases (1). Selective binding to an appropriate functional form of the receptor and timely dissociation are prerequisites for the proper function of arrestin in the cell (1, 2). Phosphorylation and activation recognition sites on arrestin bind simultaneously to the phosphorylated receptor C terminus and regions of receptor that change conformation upon activation, respectively (2). These two interactions activate arrestin, allowing its transition into a high affinity receptor-binding state (2). Several lines of evidence suggest that a major conformational rearrangement of the arrestin molecule is a part of this transition (2-5). First, arrestin binding to the receptor has a very high Arrhenius activation energy (3). Second, the susceptibility of arrestin to proteolysis upon its binding to phosphorylated and light-activated rhodopsin (P-Rh*) 1 changes dramatically (4). Third, an additional hydrophobic binding site, which is not involved in its interaction with light-activated (Rh*) or phosphorylated dark (P-Rh) rhodopsin participa...

Conformation of receptor-bound visual arrestin

“…In view of strong experimental support for one-to-one arrestin-receptor interaction, bindinginduced conformational changes in arrestin were expected to make the receptor-binding surface more compact, largely by the proposed movement of the two arrestin domains (47). This model was supported by the findings that progressive deletions in the interdomain hinge reduced the ability of all arrestins to bind receptors (32,48). However, the data presented here reveal only subtle changes in the interdomain distances, ruling out a large clam shell-like relative motion of the N and C domains (16), but a small rotation of one domain relative to the other about the long axis could occur.…”

“…1). The addition of extra residues to the hinge does not affect P-Rh* binding, whereas increasing deletions progressively reduces the ability of arrestin-1 to bind P-Rh* (32). This suggested that the transition of arrestin-1 to the active receptor-bound state requires an extended hinge, which led to the idea that the domains move relative to each other, closing in on the receptor (the "clam-shell" model) (16).…”

Functional map of arrestin-1 at single amino acid resolution

“…Release of the C tail is one of the major conformational changes occurring during arrestin activation as suggested by the sequential multisite binding model (17) and confirmed by EPR (24) and NMR (20) spectroscopic studies. The recent crystal structure of p44 arrestin-1, lacking the arrestin C tail, revealed marked conformational changes compared with a previous structure of p44 arrestin-1 (25), including a 20°rotation between the C and N domains (9) as had previously been suggested based on arrestin truncations (26) and molecular modeling (27). It is yet unclear to what extent this structure represents arrestin in the receptorbound conformation.…”

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

“…The first report suggested that in response to GPCR activation arrestin-3 binds JNK3␣2 and ASK1, whereas the recruitment of MKK4, which is necessary to make a complete signaling module, was believed to be indirect (6). Subsequent work showed that arrestin-3 promotes JNK3␣2 phosphorylation independently of GPCRs (7)(8)(9)11), in agreement with the finding that conformationally biased arrestin mutants, both the pre-activated 3A form with enhanced propensity to bind GPCRs (43-47) and the one "frozen" in basal-like conformation with impaired ability to bind receptors (11,48,49), interact with JNK3␣2 similarly to WT arrestin-3 (26,49). Arrestin-3 was also shown to co-immunoprecipitate with MKK4 essentially as efficiently as with ASK1 and JNK3␣2 (8,9,11).…”