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

FEBS Lett. 1999 Jul 23;455(3):367-71. doi: 10.1016/s0014-5793(99)00904-7.

Abstract

To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl-terminus. An approximately 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 AT1A 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 AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT1A receptor function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • CHO Cells
  • Calcium / metabolism*
  • Calmodulin / metabolism*
  • Cricetinae
  • DNA Primers / genetics
  • Feedback
  • In Vitro Techniques
  • Molecular Sequence Data
  • Rats
  • Receptor, Angiotensin, Type 1
  • Receptors, Angiotensin / chemistry*
  • Receptors, Angiotensin / genetics
  • Receptors, Angiotensin / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Calmodulin
  • DNA Primers
  • Receptor, Angiotensin, Type 1
  • Receptors, Angiotensin
  • Recombinant Fusion Proteins
  • Calcium