The microtubule-binding protein Hook3 interacts with a cytoplasmic domain of scavenger receptor A.
The class A scavenger receptor (SR-A) is a multifunctional transmembrane glycoprotein that is implicated in atherogenesis, innate immunity, and cell adhesion. Despite extensive structure-function studies of the receptor, intracellular molecules that directly interact with SR-A and regulate the receptor trafficking have not been determined. In the current study, we have identified a microtubule-binding protein, Hook3, as a novel interacting partner of SR-A. The association between a rat Hook3 isoform and SR-A was suggested by yeast two-hybrid screening and mass spectrometry analysis of SR-A-cytoplasmic domain-bound proteins in rat alveolar macrophages. The binding of overexpressed and endogenous human Hook3 to SR-A was demonstrated by pull-down assay and co-immunoprecipitations. Furthermore, endogenous murine SR-A and HK3 co-sedimented from cell lysates isolated from Raw264.7 murine macrophage cells. The interaction of Hook3 with SR-A was significantly stimulated after SR-A had recognized the extracellular ligand. Studies using truncations demonstrated that the positively charged C-terminal Val614-Ala717 region of human Hook3 was required for the interaction with the negatively charged residues, Glu12, Asp13, and Asp15 in the human SR-A cytoplasmic domain. By transfecting small interfering RNA targeting Hook3, total and surface expression, receptor-mediated ligand uptake and protein stability of SR-A were significantly promoted, whereas the protein synthesis and maturation were not altered. We propose for the first time that Hook3 may participate in the turnover of the endocytosed scavenger receptor.
Sano
Hitomi
H
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan. sanohito@sapmed.ac.jp
Ishino
Masaho
M
Krämer
Helmut
H
Shimizu
Takeyuki
T
Mitsuzawa
Hiroaki
H
Nishitani
Chiaki
C
Kuroki
Yoshio
Y
D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D051117:Scavenger Receptors, Class A / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D008024:Ligands; C495139:MSR1 protein, human; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D051117:Scavenger Receptors, Class A; C425466:hook3 protein, human