PDBsum entry 1zsh

Signaling protein

PDB id

1zsh

Contents

			Protein chain

					354 a.a.

			Ligands

					IHP

			Metals

					_MG

			Waters ×93

References listed in PDB file

Key reference

Title

Nonvisual arrestin oligomerization and cellular localization are regulated by inositol hexakisphosphate binding.

Authors

S.K.Milano, Y.M.Kim, F.P.Stefano, J.L.Benovic, C.Brenner.

Ref.

J Biol Chem, 2006, 281, 9812-9823. [DOI no: 10.1074/jbc.M512703200]

PubMed id

16439357

Abstract

Interactions between arrestins and phosphoinositides have been reported to regulate multiple membrane-associated signaling and trafficking events including clathrin-mediated endocytosis and light adaptation in Drosophila. Arrestins have been proposed to have nuclear and cytosolic functions as well, although the ligand dependence of these functions has not been investigated. Here we characterize the structural, molecular, and cellular interactions between arrestin-2 and inositol hexakisphosphate (inositol 1,2,3,4,5,6-hexakisphosphate (IP(6))). The crystal structure of the arrestin-2.IP(6) complex was solved to 2.9 A with crystal lattice contacts suggesting two sites on a protein monomer mediating IP(6) binding. Mutagenesis coupled to isothermal titration calorimetry and tritiated IP(6) binding assays confirmed two-site binding with a low affinity IP(6)-binding site in the N-domain and a high affinity site in the C-domain. Native gel electrophoresis, gel filtration, and analytical ultracentrifugation demonstrated the ability of IP(6) to promote arrestin-2 oligomerization via the two crystallographically defined ligand-binding locations. In addition, analysis in mammalian cells revealed that arrestin-2 not only undergoes homo-oligomerization, but it can also hetero-oligomerize with arrestin-3 in a manner that depends on IP(6)-binding sites. Mutation of either IP(6)-binding site in arrestin-2 disrupted oligomerization while interactions with known binding partners including clathrin, AP-2, and ERK2 were maintained. Subcellular localization studies showed that arrestin-2 oligomers are primarily cytoplasmic, whereas arrestin-2 monomers displayed increased nuclear localization. Thus, by promoting cytosolic oligomerization, IP(6) binding is proposed to be a negative regulator of interactions of arrestin with plasma membrane and nuclear signaling proteins.



	Figure 5. Arrestin-2 and -3 can homo- and heteroassociate in COS-1 cells. A, co-immunoprecipitation experiment showing wild type arrestin-2 associating with itself. Mutation of the two crystallographically defined IP[6]-binding sites on arrestin-2, either independently or combined, disrupts self-association. HA-tagged wild type and mutant arrestin-2 proteins were overexpressed in COS-1 cells with or without FLAG-tagged wild type arrestin-2. HA-arrestins were immunoprecipitated and blotted for FLAG-arrestin-2. Immunoprecipitate (IP) and lysate expression levels are shown. B and C, co-immunoprecipitation experiments showing arrestin-2 mutants effectively interacting with known binding partners. HA-tagged wild type and mutant arrestin-2 proteins were overexpressed in COS-1 cells. HA-arrestins were immunoprecipitated and blotted for clathrin, β[2]-adaptin, and ERK2. Immunoprecipitate and lysate expression levels are shown. D, co-immunoprecipitation experiment showing that wild type arrestin-2 and -3 can homo- and heteroassociate. HA-tagged arrestin-2 and -3 proteins were overexpressed in COS-1 cells with or without FLAG-tagged arrestin-2 or -3. HA-arrestins were immunoprecipitated and blotted for FLAG-arrestins. -indicates mock transfection. IB, immunoblot; WT, wild type; arr2, arrestin-2; arr3, arrestin-3.		Figure 6. Arrestin-2 oligomers are deficient in nuclear transport. HEK293 cells were transiently transfected with either wild type (WT) or mutant arrestin-2 proteins. 48 h post-transfection the cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). The slides were visualized, and images were acquired on an Olympus BX-61 fluorescence microscope.

PROCHECK

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