The molecular acrobatics of arrestin activation

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Abstract

Arrestin proteins play a key role in desensitizing G-protein-coupled receptors and re-directing their signaling to alternative pathways. The precise timing of arrestin binding to the receptor and its subsequent dissociation is ensured by its exquisite selectivity for the activated phosphorylated form of the receptor. The interaction between arrestin and the receptor involves the engagement of arrestin sensor sites that discriminate between active and inactive and phosphorylated and unphosphorylated forms of the receptor. This initial interaction is followed by a global conformational rearrangement of the arrestin molecule in the process of its transition into the high-affinity receptor-binding state that brings additional binding sites into action. In this article, we discuss the molecular mechanisms that underlie the sequential multi-site binding that ensures arrestin selectivity for the active phosphoreceptor and high fidelity of signal regulation by arrestin proteins.

Section snippets

How does arrestin know when to bind and when to dissociate?

From a biological standpoint, the timing of arrestin binding to the receptor and its subsequent dissociation is of utmost importance. Both events appear to be governed by an exquisite arrestin selectivity for the phosphorylated activated form of the receptor (P–R*). Using the visual arrestin–rhodopsin model, we found that arrestin demonstrates virtually no binding to the inactive unphosphorylated receptor (R), a low level of binding to the active unphosphorylated receptor (R*) and the

Basal arrestin conformation: the gun is cocked

Free arrestin is an elongated two-domain molecule with a ‘wingspan’ of ∼70 Å (Figure 2a) (twice the size of the cytoplasmic tip of rhodopsin, the only GPCR with a known crystal structure) 11, 12, 18. The localization of identified receptor-binding elements on the concave sides of both domains strongly suggests that the domains must move relative to each other to bring all these elements into contact with the receptor simultaneously. The extended inter-domain connector (hinge region) makes this

Arrestin activation: how does the receptor pull the trigger?

The idea that arrestin has ‘sensor’ sites was based on the arrestin selectivity profile (Figure 1a) and on the fact that short C- and N-terminal deletions yield mutants that bind well to R*, P–R* and inactive P–R (but not to R). Apparently, constraints in the arrestin molecule are necessary for its high selectivity for P–R*, whereas their release by mutagenesis allows binding to non-preferred functional forms of the receptor 16, 29. Thus, P–R* probably releases these constraints in wild-type

Arrestin meets phosphates: two-step dance

Arg175 is not exposed, being localized at the bottom of the N-domain cavity of arrestin (Figure 2f), suggesting that the phosphates must first be met by other elements and then delivered to Arg175. One striking feature of the receptor-binding side of the arrestin N-domain is the abundance of positive charges on the surface (Figure 2f). Elimination of two of these, Lys14 and Lys15, completely abolishes arrestin binding to P–R*. Even a K15A mutation alone reduces binding by >80% [14]. However,

Does the arrestin phosphate-sensing mechanism make sense?

Virtually all GPCRs have multiple (up to 15–20) phosphorylation sites localized in different intracellular elements: C-terminus (e.g. rhodopsin and β2-adrenoceptor), third cytoplasmic loop (e.g. muscarinic acetylcholine M2 receptor, α-adrenoceptors) or second cytoplasmic loop (mu opioid peptide receptor). In most cases, receptor kinases ‘hit’ these sites at random and thus an incredible variety of phosphorylated forms of the same receptor are produced. Even in rhodopsin, where the number of

Arrestin activation: local movements or global conformational change?

The idea that arrestin binding to rhodopsin involves a major conformational re-arrangement was proposed long ago and was based on the unusually high energy barrier of this interaction [36]. This hypothesis is supported by ample indirect evidence 11, 12, 13, 14, 15, 16, 22, 23, 27, 28, 29 and was one of the cornerstones of the sequential multi-site binding model [16] (Figure 1b). However, direct proof requires solution of the crystal structure of the arrestin–receptor complex, which remains

Is the activation mechanism conserved in the arrestin family?

Comparison of the structures of visual arrestin and arrestin-2 11, 12, 19, 20 reveals a striking similarity of overall fold. Detectable differences in the structures are limited to several loops where similar variations exist in different crystal forms of the same protein 11, 12. B-factor (crystallographic measure of mobility) distribution suggests that these variations reflect their high flexibility rather than arrestin subtype-specific conformation [12]. Structural similarity implies the

Does arrestin have an activation sensor?

The model of arrestin binding (Figure 1b) implies perfect symmetry (as far as arrestin is concerned) between receptor phosphorylation and activation. Arrestin is proposed to have two sensor sites, detecting the two aspects of the functional state of the receptor (i.e. phosphorylated and activated), and to work as a molecular coincidence detector, assuming the high-affinity receptor-binding conformation when both sensors are engaged simultaneously. An alternative model, where receptor-attached

Putting the pieces together: the molecular acrobatics of arrestin activation

Known ‘activating’ mutations invariably map onto the two ‘hot spots’ in the three-dimensional structure of arrestin: the polar core and the three-element interaction (Figure 3a). Both interactions keep the relative orientation of the two domains 11, 12, which suggests that in the process of arrestin activation the domains move relative to each other. The importance of the length of the inter-domain hinge [13] strongly supports this hypothesis. The current model of arrestin activation (Figure 3b

The next level of complexity: multiple active conformations?

Arrestin activation disrupts every interaction that stabilizes the basal arrestin conformation, but it does not appear to create a set of new interactions that predetermine the shape of the active state. Although β-strand ‘sandwiches’ at the core of arrestin domains probably do not change, a varying extent of domain movement and flexibility of the loops might enable arrestin to assume different active conformations, ‘molding’ itself on the interaction partner. This flexibility 11, 12, 13, 14, 15

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