Robust self-association is a common feature of mammalian visual arrestin-1

Arrestin-14 binds light-activated rhodopsin phosphorylated by GRK1 (a.k.a. rhodopsin kinase) with high affinity (1), ensuring the termination of light-induced signaling with sub-second kinetics (2). Arrestin-1 knockout in mice dramatically slows the photoresponse shutoff in rod (3) and cone (4) photoreceptors. Arrestin-1 deficiency in humans results in Oguchi disease, a form of stationary night blindness (5). Arrestin-1 is expressed at very high levels in both photoreceptor types, being the second most abundant signaling protein after corresponding opsins (4, 6, 7). Considering that the rhodopsin concentration in rod outer segments (OS) is ~3 mM (8), the average cytoplasmic concentration of arrestin-1 (which is expressed at ~0.8:1 ratio to rhodopsin (6, 7, 9)) is expected to be >1 mM (1). Dark-adapted rods are used in most studies of the molecular mechanisms of rod signaling in genetically modified mice (reviewed in (10–12)). In the dark, ~85% of arrestin-1 resides in the inner segments, cell bodies, and synaptic terminals (6, 9, 13–15), which brings its concentration in these compartments to >2 mM (9). While the majority of the functional studies were performed in mice and humans, the biochemical properties of arrestin-1 were mostly studied using bovine protein. Bovine arrestin-1 robustly self-associates (16, 17), cooperatively forming dimers and tetramers (16, 18, 19). It is unclear whether this is a peculiar property of bovine protein, or a common feature of mammalian arrestin-1 species. In addition, since only monomeric arrestin-1 binds rhodopsin and quenches the signaling (18), the concentration of the monomer is an important functional parameter; it can be calculated based on total arrestin-1 if the self-association constant(s) are known. In view of therapeutic potential of “enhanced” mutants that can compensate for deficits of rhodopsin phosphorylation in vivo (20), characterization of human arrestin-1 is particularly important. Therefore, we explored oligomerization of purified mouse and human arrestin-1 and found that self-association is a common feature of mammalian arrestin-1. Interestingly, we found that while the values of dimerization and tetramerization constants of arrestin-1 from three species are very different, the underlying molecular interactions appear to be similar: the same point mutations render bovine and mouse arrestin-1 constitutively monomeric.