Chapter Three - Structural Determinants of Arrestin Functions
Introduction
As far as size is concerned, arrestins are quite average, 44–48 kDa soluble proteins. Functionally, however, arrestins are far from being average in many ways, demonstrating that evolution can pack incredible versatility into ~ 400 amino acids.
The discovery of the first member of the arrestin family (modern systematic name arrestin-1a) was rather unremarkable, except that it was discovered twice: first as S-antigen, the target of auto-antibodies in uveitis,1 then as a 48-kDa protein that binds light-activated rhodopsin,2 preferring the phosphorylated form.3 Eventually it became clear that both are the same protein, which prevents G protein activation by light-activated phosphorylated rhodopsin,4 thereby blocking (arresting) further signaling.
All this happened before the seminal discovery of striking similarity in sequence and topology between the β2-adrenergic receptor (β2AR) and rhodopsin,5 which led to the concept that there is a large family of G-protein-coupled receptors (GPCRs; also known as seven transmembrane domain receptors or 7TMRs) and fruitful ideas regarding the similarity of signaling and regulatory mechanisms in this family. The first nonvisual arrestin, termed β-arrestin because of its preference for the β2AR over rhodopsin, was cloned soon thereafter,6 followed by another nonvisual subtype (termed β-arrestin2,7 arrestin-3,8 and hTHY-ARRX,9 respectively) and cone-specific arrestin.10, 11 Considering that different vertebrate species express from 800 to > 3400 distinct GPCRs (SEVENS database; http://sevens.cbrc.jp/), the fact that we only have four arrestin subtypes12 is rather remarkable. Moreover, arrestin-1 and -4 are largely restricted to photoreceptors,13 whereas the two nonvisual subtypes are ubiquitously expressed and interact with hundreds of different GPCRs.12, 14 Even this striking versatility is only half of the story—in addition to receptors, arrestins bind dozens,12 and possibly hundreds,15 of amazingly diverse proteins, serving as multifunctional signaling organizers in the cell (see Chapter 1).
Section snippets
What the Crystal Structure Reveals, and What It Does Not
Visual arrestin-1 was the first subtype discovered,2 functionally characterized,4 cloned,16 and crystallized.17, 18 The structure revealed a unique fold: an elongated molecule consisting of two cup-like domains with similar cores, each organized as a seven-strand β-sandwich (Fig. 3.1). Subsequently solved structures of arrestin-2,19, 21 arrestin-3,20 and arrestin-422 and even the short splice variant of arrestin-123 showed rather disappointing similarity, offering surprisingly few clues
How Do Arrestins Fit Receptors?
There is an obvious caveat in fitting known arrestin and receptor structures: for the complex to form, both arrestin and receptor must be in an active conformation, and the receptor also must be phosphorylated.14, 30 While the effect of receptor-attached phosphates on its conformational state is completely unknown, activation-induced changes were well characterized, first by a series of site-directed spin-labeling studies of rhodopsin55, 56, 57, 58, 59 then by the solution of several crystal
Interactions with Other Signaling Proteins
Clathrin was the first nonreceptor-binding partner of arrestin proteins identified.145 Since then arrestins were shown to interact with an amazing variety of trafficking and signaling proteins (see Chapter 1). The molecular mechanisms of most of these interactions remain to be elucidated. By comparison, it appears that we know a lot about the mechanics of receptor binding, although even in this area there are more questions than answers.
Designing Signaling-Biased Arrestin Mutants
To a certain extent, the natural structural organization of arrestins makes targeting different aspects of their function easier. The concave sides of both domains contain allknown receptor-binding residues (Section 3.3), most other partners interact with elements on the other side of the molecule that remain accessible in the arrestin–receptor complex, whereas clathrin and AP2 engage distinct sites on the arrestin C-tail (Section 4.1) that do not appear to overlap with the binding sites of any
Conclusions: Where Do We Go from Here?
It might appear that as proteins go, arrestins are fairly well studied structurally and functionally. It is clear that members of this small protein family, which apparently emerged relatively late in evolution,12 likely after GPCR kinases,176 serve as multifunctional signaling organizers in the cell. However, while known phenomenology is rich and pretty well described,177 the structural basis of most arrestin functions remains obscure. The molecular mechanisms underlying arrestin interactions
Acknowledgments
The authors are grateful to our collaborators, whose expertise and efforts made many of the studies discussed here possible. Supported by NIH Grants EY011500, GM077561, GM081756 (VVG), NS065868, and DA030103 (EVG).
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