Abstract
In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate. This peptide also affected the specific binding of [3H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5'-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs.
Highlights
Synthesis of i3 cyc—To synthesize i3 cyc, we used a r-V2 receptor (V2-R) model derived from the rhodopsin coordinates (Protein Data Bank accession number 1F88) to select the appropriate N- and C-terminal residues at the cytoplasmic ends of TMs 5 and 6, i3 Peptide Acts on Vasopressin V2 Receptor Dimer respectively
The N- and C-terminal residues (Gln225 and Leu274) of the i3 loop were replaced with Gly and S-carboxymethyl cysteine, respectively, to reproduce a distance similar to that evaluated for the r-V2-R model (Fig. 1)
To test the hypothesis that a peptide corresponding to V2-R third intracellular loop could affect the receptor dimer structural arrangement and function, we synthesized a cyclic peptide of 51 residues mimicking the i3 loop structure (i3 cyc)
Summary
Fmoc-Rink amide and pseudo-proline dipeptide building blocks were obtained from NovaBiochem (Laufelfingen, Switzerland). Tritiated [Arg8]-vasopressin (60 Ci/mmol) ([3H]AVP) was obtained from PerkinElmer Life and Analytical Sciences. [125I]CGP64213 was synthesized from ethyl-(1,1diethoxy-ethyl) phosphonate as described elsewhere [23] and labeled to a specific radioactivity of Ͼ2000 Ci/mmol (ANAWA AG, Wangen, Switzerland). [125I](Ϫ)-Iodocyanopindolol ([125I]CYP), [␣-32P]ATP (30 Ci/mmol), and [3H]cAMP (32 Ci/mmol) were from PerkinElmer Life Sciences Tritiated [Arg8]-vasopressin (60 Ci/mmol) ([3H]AVP) was obtained from PerkinElmer Life and Analytical Sciences. [Arg8]-vasopressin was obtained from Bachem. [125I]CGP64213 was synthesized from ethyl-(1,1diethoxy-ethyl) phosphonate as described elsewhere [23] and labeled to a specific radioactivity of Ͼ2000 Ci/mmol (ANAWA AG, Wangen, Switzerland). [125I](Ϫ)-Iodocyanopindolol ([125I]CYP), [␣-32P]ATP (30 Ci/mmol), and [3H]cAMP (32 Ci/mmol) were from PerkinElmer Life Sciences
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