title: Two distinct calmodulin binding sites in the third intracellular loop and carboxyl tail of angiotensin II (AT(1A)) receptor.
abstract: In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT(1A)), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214-231) and carboxyl tail of the receptor (ct, 302-317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT(1A) holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca²⁺-dependent fashion. The former is a 1-12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein βγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gβγ binding, but reduced the ability of calmodulin to compete with Gβγ, suggesting that calmodulin and Gβγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gβγ binding to i3 and ct regions of the AT(1A) receptor strongly suggests that calmodulin plays critical roles in regulating Gβγ-dependent signaling of the receptor.
authors.affiliation: Department of Pathogenobiology, Bethune College of Medicine, Jilin University, Changchun, Jilin, P. R. China.
authors.forename: Renwen
authors.initials: R
authors.lastname: Zhang
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Liu
authors.forename: Youxing
authors.initials: Y
authors.lastname: Qu
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; D021122:Protein Subunits; D044140:Receptor, Angiotensin, Type 1