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

Abstract

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.

Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

“…Y2H assay was performed as described above [20, 21]. Briefly, the pGBKT7 plasmids containing the full-length Ca v β 1a cDNA were introduced into the Y2H gold yeast strain using a modified lithium-acetate protocol, and the transformants were selected on SD/-Trp plates.…”

Protein-Protein Interactions at the Adrenergic Receptors

“…Another study reported that mammalian tolloid (mTLD) could interact with the α 1a AR [48]. The CUB5 domain of mTLD, a zinc-finger matrix metalloprotease of the astacin family, interacted with α 1a AR C-tail in a yeast two hybrid screen.…”

The α1-adrenergic receptors: diversity of signaling networks and regulation

“…Another study reported that the CUB5 domain of mammalian tolloid (mTLD), a zinc-finger matrix metal-loprotease of the astacin family, interacted with α 1a -AR C-tail in a yeast two hybrid screen (37). Overexpression of mTLD reduced the number of cell surface receptors without affecting total receptor level or affinity when transiently expressed in HEK293 cells.…”