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 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.
Xu
Qi
Q
Institute of Vascular Medicine, Peking University Third Hospital, No.49 Huayuan North Road, Haidian District, Beijing, P.R. China 100083.
Xu
Ning
N
Zhang
Tan
T
Zhang
Hui
H
Li
Zijian
Z
Yin
Feng
F
Lu
Zhizhen
Z
Han
Qide
Q
Zhang
Youyi
Y
D000595:Amino Acid Sequence; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000737:chemistry / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D008666:Metalloendopeptidases / Q000502:physiology; D045726:Metalloproteases / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D018340:Receptors, Adrenergic, alpha-1 / Q000032:analysis / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D055394:Tolloid-Like Metalloproteinases; D020798:Two-Hybrid System Techniques
C547713:ADRA1A protein, human; D019485:Bone Morphogenetic Proteins; D018340:Receptors, Adrenergic, alpha-1; D045726:Metalloproteases; D055394:Tolloid-Like Metalloproteinases; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1; D002118:Calcium