Arrestins are multi-functional regulators of cellular signaling capable of interacting with more than 20 partners, often binding 2–3 different proteins simultaneously 3
. Two interconnected aspects of arrestin function are the regulation of the activity of its partners and their localization to a particular intracellular compartment. The first arrestin targets discovered, GPCRs, are restricted to the plasma membrane and endocytic vesicles. Therefore the interaction of numerous proteins (e.g., Src, JNK3, ERK1/2, Mdm2, etc) with receptor-bound arrestin localizes these molecules to receptor-rich membranes 4
. Here we describe a novel arrestin binding partner with a clearly defined cellular localization, microtubules, and demonstrate that the arrestin-MT interaction serves to localize certain signaling molecules to the cytoskeleton. Arrestin-dependent recruitment of protein kinase ERK and ubiquitin ligase Mdm2 to the microtubules differentially affects their activity, silencing ERK and directing Mdm2 to cytoskeletal substrates.
Our recent discovery of the light-regulated interaction of arrestin with MTs in rod photoreceptors 11
prompted us to test whether the other three members of the mammalian arrestin family interact with the cytoskeleton. We found that in intact cells and in vitro all four arrestin subtypes bind microtubules (, , ). The affinity of arrestin2 for MTs (KD
~26 μM; ) is comparable to the normal tubulin concentration in many cell types 46
, which reaches 150 μM in mature neurons 46
, suggesting that this interaction occurs in vivo
. Indeed, our data show that an appreciable proportion of non-visual arrestins is bound to microtubules in cells (). Interestingly, several other regulators of GPCR signaling, such as G proteins 48
and GRK2 49
also associate with microtubules. Considering the number of experimental studies of arrestins and microtubules, one might wonder why the connection between the two was not noticed before. In fact, both non-visual arrestins were previously found in the detergent-insoluble fraction 51
, but the authors did not attach much significance to this observation.
Conceivably, arrestin association with MTs may serve three functions (which are not mutually exclusive): 1) to keep arrestins away from receptors, similar to its apparent role in rod photoreceptors 11
; 2) to sequester arrestin binding partners to regulate their activation; 3) to mobilize signaling proteins to the cytoskeleton and direct their activity toward MT-associated substrates. The role of this interaction depends on the functional capabilities of MT-bound arrestin, and in particular, on the accessibility of the sites used by its other binding partners. The receptor binding site was mapped by numerous studies 22
to the concave sides of both arrestin domains. The localization of the interaction sites for the non-receptor partners on the other side of the molecule allows arrestin to mobilize various signaling proteins to the receptor 4
. Our data show that microtubules bind to the same arrestin surface as the receptor (, )9
, suggesting that the elements involved in the interactions with the non-receptor partners should be accessible in the MT-bound form. Indeed, our finding that arrestin mobilizes protein kinase ERK1/2 and ubiquitin ligase Mdm2 to the cytoskeleton (, ) clearly shows that this is the case. However, the functional capabilities of MT- and receptor-bound arrestin are different: arrestin mobilizes ERK, Mdm2, but not c-Raf-1, MEK1, JNK3, or PP2A to microtubules, whereas all of these partners are recruited to the arrestin-receptor complex.
Receptor binding induces a global conformational change in arrestin 2
that is widely believed to facilitate the binding of clathrin, AP2, Src, MAP kinases, and other proteins to the complex 4
. The deletions in the inter-domain hinge impede arrestin transition into this active conformation, thereby dramatically reducing arrestin binding to the receptor () 13
. In contrast, hinge deletions actually enhance arrestin binding to MTs, suggesting that the conformations of MT-bound and receptor-bound arrestins are different (). Not surprisingly, the functional consequences of arrestin-dependent ERK mobilization to the MTs is opposite to its recruitment to GPCRs. Receptor-bound arrestin facilitates ERK activation 39
, whereas cytoskeletal localization of ERK by arrestin keeps it inactive, likely because arrestins do not mobilize the upstream kinases c-Raf-1 and MEK1 to microtubules (). Conversely, the result of arrestin-dependent recruitment of Mdm2 to GPCRs and MTs is similar: Mdm2 ubiquitinates the receptor in one case 44
and cytoskeletal proteins in the other (). Because arrestins bind another ubiquitin ligase, TRAF6 54
, we cannot exclude the possibility that the mobilization of other ubiquitin ligases is partially responsible for the arrestin-dependent increase in the ubiquitination of cytoskeletal proteins. However, close correlation between arrestin-dependent Mdm2 mobilization and ubiquitination level in the MT pellet () suggests that Mdm2 plays an important role. Thus, by recruiting Mdm2 to microtubules arrestin redirects its activity to a different set of substrates. Interestingly, rod arrestin successfully recruits Mdm2 to MTs and facilitates the ubiquitination of microtubule-associated proteins to the greatest extent (), suggesting that its association with the cytoskeleton may have earlier unappreciated functions in photoreceptors. Rod arrestin also enhances the ubiquitination of soluble proteins (), apparently by stabilizing Mdm2 in the cell.
The binding of arrestin proteins to microtubules with affinities that ensure their partial co-localization with the cytoskeleton in intact cells () is a novel link in the network of cellular signaling pathways. Accumulating evidence shows that a number of signaling proteins that were believed to selectively interact with receptor-bound arrestin actually bind free arrestin in the cytoplasm 42
and the microtubule-bound form (, ). Notably, two out of the six arrestin partners tested in our study are recruited to microtubules in an arrestin-dependent fashion. It is tempting to speculate that a number of other known binding partners may also be mobilized to the cytoskeleton via an arrestin-dependent mechanism with significant functional consequences. The recruitment of signaling molecules may affect their activation state (, ) and/or microtubule dynamics which is known to be regulated by post-translational modifications of tubulin and associated proteins 34
. The full range of biological implications of the functional link between arrestins and the cytoskeleton remains to be elucidated.