Linear V1-vascular vasopressin antagonists suitable for radioiodination, biotinylation, and fluorescent labeling

Thibonnier, Marc; Bayer, A. L.; Madhun, Zuhayr T.

doi:10.1152/ajpendo.1993.265.6.e906

Cited by 4 publications

(2 citation statements)

References 9 publications

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“…The sustained activity observed could be due to the following: 1) the receptors present on the cell surface, or 2) to sustained activity of the internalized receptor-G-protein complex. To distinguish between these possibilities, the effect of the V1aR antagonist TyrPhaar in the accumulation of IPs was tested (19). Four minutes after addition of 100 nM AVP, the hormone was removed and replaced by 1 M TyrPhaar, and the effect of the antagonist on PLC activity was measured at different times.…”

Section: Phosphorylation Of the V1amentioning

confidence: 99%

Transient Phosphorylation of the V1a Vasopressin Receptor

Innamorati¹,

Sadeghi²,

Birnbaumer³

1998

Journal of Biological Chemistry

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The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.Agonist binding and activation of many G-protein-coupled receptors is followed by desensitization, an attenuation of the cellular response to the ligand that prevents sustained stimulation. Studies with rhodopsin and the ␤2-adrenergic receptor have implicated phosphorylation in receptor desensitization (1, 2) Receptor phosphorylation promotes the binding of arrestin, which in turn uncouples the receptor from the G-protein and enhances sequestration. In the particular case of the ␤2-adrenergic receptor, it has been postulated that sequestration of the phosphorylated receptor facilitates cleavage of the phosphate groups by phosphatases that associate with the endosomes and allows the recycling of the de-phosphorylated active protein to the cell surface (3). Data obtained with other G-protein-coupled receptors have indicated that phosphorylation is not required to observe receptor sequestration and desensitization. For example, a truncated form of the V2 vasopressin receptor lacking the phosphorylation acceptor sites at the carboxyl-terminal end was sequestered and desensitized, although to a lesser extent than the wild type receptor (4). Similarly, a mutant angiotensin II receptor missing a portion of the carboxyl terminus did not exhibit ligand-induced sequestration although it contained a putative GRK...

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Section: Phosphorylation Of the V1amentioning

confidence: 99%

Transient Phosphorylation of the V1a Vasopressin Receptor

Innamorati¹,

Sadeghi²,

Birnbaumer³

1998

Journal of Biological Chemistry

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“…The current report is the first characterization of the iodinated peptide. The apparent K d value of 0.168 nM for iodinated antagonist-liver membrane interactions is approximately one third greater than the value (0.06 nM) observed for the analogue containing an arginine at position 6 instead of a lysine (10) and ϳ1 order of magnitude less than an analogue containing valine in position 4 instead of glutamine and having a Tyr-NH 2 carboxylterminal residue at position 8 (18). Aside from the obvious advantages of having a highly selective and high affinity antagonist that can be radioiodinated for use with small amounts of tissue or for autoradiographic studies, the lysine residue at position 6 allows the formation of derivatives for photoaffinity and fluorescent labeling and for biotinylation.…”

Section: Discussionmentioning

confidence: 67%

A New Linear V_1AVasopressin Antagonist and Its Use in Characterizing Receptor/G Protein Interactions

Straková¹,

Kumar²,

Aj³

et al. 1997

Mol Pharmacol

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We characterized a new iodinated, high affinity, linear V1a vasopressin antagonist, phenylacetylD-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH2. The antagonist bound specifically to the V1a vasopressin receptor in crude rat liver membranes with an apparent Kd value of 0.168 nM. This affinity is approximately 1 order of magnitude greater than that of the natural agonist, vasopressin. The inhibitory activity of the antagonist can be demonstrated by its inability to elicit activation and uncoupling of G proteins from the receptor. Thus, after occupancy of receptor sites in rat liver membranes with labeled antagonist and detergent solubilization, the labeled receptor (approximately 60 kDa) was eluted as a stable 400-kDa complex on size-exclusion chromatography. In contrast, when the receptor sites were occupied by the agonist [3H]vasopressin, the receptor eluted as a 60-kDa peak. Coincubation of membranes with iodinated antagonist and an excess of unlabeled vasopressin caused both reduced antagonist binding and a complete shift from the 400-kDa to the 60-kDa peak. The addition of vasopressin to unliganded 400-kDa fractions resulted in a 75% increase in [35S]guanosine-5'-O-(3-thio)triphosphate binding activity, indicating that the 400-kDa fraction contains complexes between the V1a receptor and G proteins. The vasopressin-elicited increase was inhibited by antagonist. Using specific antibodies and immunoadsorption to protein A/Sepharose columns, we found that G protein isotypes G(alpha q/11), G(alpha i3), and G(alpha s), and effector enzymes PLC-beta1, PLC-gamma2 and PLA-2 were associated with the antagonist-labeled receptor in the 400-kDa fraction. Because the 400-kDa complex was found in the absence of ligand, the V1a receptor and the appropriate G proteins and effector enzymes are likely preassociated with each other and do not aggregate after antagonist addition. The association of V1a receptor with the different specific G proteins and effector enzymes is consistent with the multiple actions of vasopressin on liver cells. Antibodies directed against a portion of the carboxyl-terminal domain of the V1a receptor interacted with 60-kDa antagonist-occupied receptor but not with receptor in the 400-kDa complex. These results suggest that the carboxyl-terminal region of the receptor is sterically hindered when coupled to G proteins. The iodinated linear vasopressin antagonist therefore allows stable receptor/G protein complexes and can be an important tool (along with the antisera) for use in the study of factors that control V1a receptor/G protein coupling.

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Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents☆

Manning

Stoev

Chini

et al. 2008

Progress in Brain Research

256

232

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Linear V1-vascular vasopressin antagonists suitable for radioiodination, biotinylation, and fluorescent labeling

Cited by 4 publications

References 9 publications

Transient Phosphorylation of the V1a Vasopressin Receptor

Transient Phosphorylation of the V1a Vasopressin Receptor

A New Linear V_1AVasopressin Antagonist and Its Use in Characterizing Receptor/G Protein Interactions

Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents☆

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Linear V1-vascular vasopressin antagonists suitable for radioiodination, biotinylation, and fluorescent labeling

Cited by 4 publications

References 9 publications

Transient Phosphorylation of the V1a Vasopressin Receptor

Transient Phosphorylation of the V1a Vasopressin Receptor

A New Linear V1AVasopressin Antagonist and Its Use in Characterizing Receptor/G Protein Interactions

Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents☆

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A New Linear V_1AVasopressin Antagonist and Its Use in Characterizing Receptor/G Protein Interactions