Identification of a Ca 2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT 1A) receptor

Abstract

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 Ca 2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca 2+/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 Ca 2+-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 Ca 2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT 1A receptor function.