Identification of a Ca2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT1A) receptor.
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.
Thomas
W G
WG
Molecular Endocrinology Laboratory, Baker Medical Research Institute, Melbourne, Australia. walter.thomas@baker.edu.au
Pipolo
L
L
Qian
H
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D005246:Feedback; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D017931:DNA Primers; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; D002118:Calcium