title: Structure of the third intracellular loop of the vasopressin V2 receptor and conformational changes upon binding to gC1qR.
abstract: The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GalphaS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted alpha-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GalphaS. Moreover, we could estimate the apparent K(d) of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.
authors.affiliation: INSERM U554, Montpellier, France.
authors.forename: Gaëtan
authors.initials: G
authors.lastname: Bellot
authors.forename: Sébastien
authors.initials: S
authors.lastname: Granier
authors.forename: William
authors.initials: W
authors.lastname: Bourguet
authors.forename: René
authors.initials: R
authors.lastname: Seyer
authors.forename: Rita
authors.initials: R
authors.lastname: Rahmeh
authors.forename: Bernard
authors.initials: B
authors.lastname: Mouillac
authors.forename: Robert
authors.initials: R
authors.lastname: Pascal
authors.forename: Christiane
authors.initials: C
authors.lastname: Mendre
authors.forename: Hélène
authors.initials: H
authors.lastname: Déméné
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005454:Fluorescence Polarization; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011951:Receptors, Complement / Q000378:metabolism*; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D011951:Receptors, Complement; D017483:Receptors, Vasopressin; C032402:complement 1q receptor