Abstract
Purpose::
Arrestins bind activated phosphorylated G protein-coupled receptors. Receptor-bound non-visual arrestins interact with numerous partners and redirect the signaling to G protein-independent pathways. Free rod arrestin robustly binds protein kinase JNK3 and E3 ubiquitin ligase Mdm2. Here we examined the mechanism of interaction of cone arrestin with JNK3 and Mdm2.
Methods::
Cone arrestin is excluded from the nuclei of photoreceptors and HEK cells. JNK3 and Mdm2 localize primarily to the nucleus. We used the ability of arrestins to redistribute their partners to the cytoplasm to study the interaction of cone arrestin with these two proteins. Tagged and untagged wild-type cone arrestin was expressed in HEK-293A cells with or without GFP-JNK3 and GFP-Mdm2. Cells expressing GFP-JNK3 and GFP-Mdm2 alone served as controls. Different mutant forms of cone and rod arrestin with increased or reduced structural flexibility and individual N- and C- domains were coexpressed with JNK3 and Mdm2 and compared to wild type proteins.
Results::
Coexpression of cone arrestin with JNK3 and Mdm2 results in the relocalization of these two proteins from the nucleus to the cytoplasm. Leptomycin B (LMB) is a potent inhibitor of protein export from the nucleus. However, the ability of cone arrestin (untagged and flag-tagged) to exclude JNK3 and Mdm2 from the nucleus is LMB-insensitive. Wild-type cone arrestin, "pre-activated" 3A and truncated mutants, and arrestin "frozen" in the basal state by hinge deletion relocalize JNK3 equally well, whereas the redistribution of Mdm2 is conformation-dependent: the mutant frozen in its basal state is the most effective. Separate N- and C-domains of both rod and cone arrestins can move JNK3 to the cytoplasm as efficiently as full-length proteins. N-domain (but not C-domain) of rod arrestin moves Mdm2. Neither domain of cone arrestin has this effect.
Conclusions::
The ability of cone arrestin to redistribute JNK3 and Mdm2 suggests that free arrestin interacts with these partners. Cone arrestin mutant "frozen" in basal conformation shows the highest ability to bind Mdm2, whereas there is no conformational preference for JNK3 binding. Binding site for JNK3 includes elements in both domains, with the affinity of partial sites on each domain sufficient for JNK3 relocalization. Mdm2 also interacts with both domains, although the main site is localized on the N domain. Thus, all four mammalian arrestins in their basal conformation bind JNK3 and Mdm2. Relevant binding sites are fairly extensive and include elements of both domains.
Keywords: photoreceptors • protein structure/function • signal transduction