Mammalian tolloid alters subcellular localization, internalization, and signaling of alpha(1a)-adrenergic receptors

In the present study, we identified the CUB5 domain of mammalian Tolloid (mTLD) as a novel protein binding to alpha(1A)-adrenergic receptor (AR) using the yeast two-hybrid system. Whereas CUB5 did not couple to either alpha(1B)-AR or alpha(1D)-AR. It was determined that amino acids 322 to 359 of alp...

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Published in:Molecular pharmacology 2006-08, Vol.70 (2), p.532-541
Main Authors: Xu, Qi, Xu, Ning, Zhang, Tan, Zhang, Hui, Li, Zijian, Yin, Feng, Lu, Zhizhen, Han, Qide, Zhang, Youyi
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bone Morphogenetic Protein 1
Bone Morphogenetic Proteins - chemistry
Bone Morphogenetic Proteins - physiology
Calcium - metabolism
Cell Line
Humans
Metalloendopeptidases - physiology
Metalloproteases - chemistry
Metalloproteases - physiology
Molecular Sequence Data
Receptors, Adrenergic, alpha-1 - analysis
Receptors, Adrenergic, alpha-1 - physiology
Signal Transduction - physiology
Tolloid-Like Metalloproteinases
Two-Hybrid System Techniques
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Summary:In the present study, we identified the CUB5 domain of mammalian Tolloid (mTLD) as a novel protein binding to alpha(1A)-adrenergic receptor (AR) using the yeast two-hybrid system. Whereas CUB5 did not couple to either alpha(1B)-AR or alpha(1D)-AR. It was determined that amino acids 322 to 359 of alpha(1A)-AR were the major binding region for CUB5. The direct interaction between alpha(1A)-AR cytoplasmic tail and CUB5 was discovered by glutathione S-transferase pull-down assay. We confirmed the interaction of mTLD with alpha(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 alpha(1A)-AR, it significantly altered the subcellular localization of the receptors. Moreover, mTLD reduced the level of cell surface alpha(1A)-ARs, delayed the initial rate of agonist-induced receptor internalization, and facilitated agonist-induced calcium transient. We have demonstrated that mTLD interacts with alpha(1A)-AR directly, alters the subcellular localization of receptor, and influences agonist-induced alpha(1A)-AR internalization and calcium signaling.
ISSN:0026-895X
DOI:10.1124/mol.105.016451