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Mammalian Tolloid Alters Subcellular Localization, Internalization, and Signaling of _1a-Adrenergic Receptors

Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China

In the present study, we identified the CUB5 domain of mammalian Tolloid (mTLD) as a novel protein binding to _1A-adrenergic receptor (AR) using the yeast two-hybrid system. Whereas CUB5 did not couple to either _1B-AR or _1D-AR. It was determined that amino acids 322 to 359 of _1A-AR were the major binding region for CUB5. The direct interaction between _1A-AR cytoplasmic tail and CUB5 was discovered by glutathione S-transferase pull-down assay. We confirmed the interaction of mTLD with _1A-AR in human embryonic kidney (HEK) 293 cells by immunoprecipitation, immunofluorescence, and fluorescence resonance energy transfer. Although mTLD did not affect the density and affinity of receptors in crudely prepared membranes from HEK293 cells stably expressing _1A-AR, it significantly altered the subcellular localization of the receptors. Moreover, mTLD reduced the level of cell surface _1A-ARs, delayed the initial rate of agonist-induced receptor internalization, and facilitated agonist-induced calcium transient. We have demonstrated that mTLD interacts with _1A-AR directly, alters the subcellular localization of receptor, and influences agonist-induced _1A-AR internalization and calcium signaling.

Address correspondence to: Youyi Zhang, Institute of Vascular Medicine, Peking University Third Hospital, No.49 Huayuan North Road, Haidian District, Beijing, P.R. China 100083. E-mail: zhangyy{at}bjmu.edu.cn