Visual Arrestin Binding to Microtubules Involves a Distinct Conformational Change

Abstract

Recently we found that visual arrestin binds microtubules and that this interaction plays an important role in arrestin localization in photoreceptor cells. Here we use site-directed mutagenesis and spin labeling to explore the molecular mechanism of this novel regulatory interaction. The microtubule binding site maps to the concave sides of the two arrestin domains, overlapping with the rhodopsin binding site, which makes arrestin interactions with rhodopsin and microtubules mutually exclusive. Arrestin interaction with microtubules is enhanced by several "activating mutations" and involves multiple positive charges and hydrophobic elements. Arrestins are soluble cytoplasmic proteins that play a critical role in the regulation of signaling by the majority of G protein-coupled receptors. Vertebrates have four different arrestin subtypes, two of which regulate rhodopsin and cone opsins in rod and cone photoreceptors, respectively, whereas two non-visual arrestins are ubiquitously expressed and regulate hundreds of different G protein-coupled receptors. All arrestins preferentially bind to the activated phosphorylated forms of their cognate receptors, shutting off G protein-mediated signaling (reviewed in Ref. 1). Non-visual arrestins also interact with numerous non-receptor binding partners, orchestrating intracellular trafficking of the arrestin-receptor complex and redirecting receptor-initiated signaling to alternative G protein-independent pathways (reviewed in Refs. 2 and 3). Recently, we identified microtubules (MTs) 4 as an interaction partner of visual (rod) arrestin (4). The difference in MT binding affinity between the two splice variants of visual arrestin expressed in bovine rods (5) determines their differential subcellular localization (4). Moreover, microtubules serve as a "default" arrestin binding partner in darkadapted rod photoreceptors (6). Dynamic interactions with rhodopsin in the light and MTs in the dark underlie the massive light-dependent translocation of rod arrestin between the inner and outer segments of photoreceptor cells (6). Here we explore the molecular mechanism of visual arrestin binding to MTs and identify arrestin elements involved in this interaction. Several lines of evidence also suggest that the conformation of microtubule-bound arrestin is different from both free and receptor-bound forms.

Opposing Effects of Inositol Hexakisphosphate on Rod Arrestin and Arrestin2 Self-Association

“…GPCRs (46)(47)(48), microtubules (14,15), and calmodulin (13) all bind to the same arrestin surface, suggesting that these partners could compete with IP6. The affinity of arrestins for their cognate receptors is subnanomolar (49,50), so that IP6 even at 100 μM does not significantly inhibit receptor binding (16,40).…”

“…The affinity of arrestins for their cognate receptors is subnanomolar (49,50), so that IP6 even at 100 μM does not significantly inhibit receptor binding (16,40). On the other hand, arrestin affinity for microtubules and calmodulin is in the micromolar range (13)(14)(15), so that IP6 may regulate its interaction with these partners. In summary, although IP6 has the potential to facilitate the selfassociation of non-visual arrestins under special circumstances in cellular compartments where these proteins are highly concentrated, it may also regulate other functions of all arrestin proteins, such as their interactions with the cytoskeleton and/or with calmodulin, the most ubiquitous regulator of Ca 2+ -dependent processes in the cell.…”

Arrestins and two receptor kinases are upregulated in Parkinson's disease with dementia

“…GRK2 and GRK5 bind microtubules and phosphorylate tubulin, possibly promoting microtubule assembly [20]. Visual arrestin has been shown to bind microtubules [38], and this ability plays a role in arrestin-mediated visual adaptation [77]. Non-visual arrestins also bind microtubules, demonstrating even higher affinity [39].…”

“…Visual arrestin has been shown to bind microtubules [38], and this ability plays a role in arrestin-mediated visual adaptation [77]. Non-visual arrestins also bind microtubules, demonstrating even higher affinity [39]. It is conceivable that coordinated action of arrestins and GRKs at microtubules is important for function of neuronal cytoskeleton, and that it may be altered by enhanced expression of arrestins and GRKs in PD patients with dementia.…”

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

“…Such conformational heterogeneity may arise from slowly exchanging conformations of the protein or/and multiple orientations of the spin-label in a given protein conformation. Determining the relative contribution of the two scenarios is not trivial and may require the use of alternate spin-label derivatives, or changing experimental temperature, 64 pressure, 65 field strengths/microwave frequency, 66 or by osmolyte perturbation. 67 Furthermore, DEER measurements in liposomes may be limited by a number of factors, including lower sensitivity, higher background contribution resulting from enhanced intermolecular interactions, and a reduction in the measurable distance range (<50Å).…”