Mammalian Tolloid Alters Subcellular Localization, Internalization, and Signaling of α1a-Adrenergic Receptors

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.