Identification of a Ca2+/calmodulin‐binding domain within the carboxyl‐terminus of the angiotensin II (AT1A) receptor

FEBS Letters

Pipolo, Luisa¹  Qian, Hongwei¹  Thomas, Walter G.¹ 

[1] Molecular Endocrinology Laboratory, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, 8008, Australia

关键词: Calmodulin-binding protein;    Receptor;    Angiotensin;    AT1A receptor;    AT1;    AT2;    AT1A;    AT1B;    types and subtypes of angiotensin II receptors;    AngII;    angiotensin II;    G-protein;    guanyl nucleotide-binding protein;    GPCR;    G-protein-coupled receptor;    IP3;    inositol(1;    4;    5)trisphosphate;    CaM;    calmodulin;    HBSS;    Hank's buffered salt solution;    CHO-K1;    Chinese hamster ovary cells;    MBP;    maltose-binding proteins;    MBP-AT1ACT;    MBP fusion protein containing the AT1A carboxyl-terminus;   

DOI  :  10.1016/S0014-5793(99)00904-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

To identify regulators of the type 1A angiotensin II receptor (AT_1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT_1A receptor carboxyl-terminus. An ∼20 kDa cytoplasmic protein interacted with the fusion protein in a Ca²⁺-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca²⁺/calmodulin and a peptide corresponding to a membrane proximal portion of the AT_1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca²⁺-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT_1A receptor by Ca²⁺/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT_1A receptor function.

【 授权许可】

Unknown