title: Association of adaptor protein TRIP8b with clathrin.
abstract: TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.
authors.affiliation: Laboratory of Receptor Cell Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, 117997 Moscow, Russia.
authors.forename: Nadezhda V
authors.initials: NV
authors.lastname: Popova
authors.forename: Igor E
authors.initials: IE
authors.lastname: Deyev
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Petrenko
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002966:Clathrin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons / Q000235:genetics; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D015221:Potassium Channels / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D002966:Clathrin; D054815:Cyclic Nucleotide-Gated Cation Channels; C578667:Hcn1 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008565:Membrane Proteins; C523657:Pex5l protein, rat; D015221:Potassium Channels; D004247:DNA