Processing and Ligand-Induced Modifications of the V2 Vasopressin Receptor

Sadeghi, Hamid Mirmohammad; Innamorati, Giulio; Esqueda, Evette E.; Birnbaumer, Mariel

doi:10.1007/978-1-4615-4871-3_43

Cited by 8 publications

(5 citation statements)

References 16 publications

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“…An alternative explanation is that phosphorylation by PKA is a prerequisite for phosphorylation by GRK, so that preventing the former also prevents the latter. This could be similar to the PKA-induced enhancement of ␤ 2 -adrenergic receptor phosphorylation by GRK (Moffett et al, 2001), but would also suggest that agonist-induced phosphorylation is not necessary for D 1 receptor desensitization, as has been suggested for several other GPCR subtypes (Malecz et al, 1998;Sadeghi et al, 1998;Olivares-Reyes et al, 2001). Finally, as noted above, the particular characteristics of desensitization observed in the present studies might have precluded detection of an effect of PKA inhibition on D 1 receptor desensitization.…”

Section: Discussionsupporting

confidence: 59%

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Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

2002

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The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D 1 receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D 1 receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D 1 -EGFP). D 1 -EGFP was similar to the untagged D 1 receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D 1 -EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D 1 receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D 1 receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.

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Section: Discussionsupporting

confidence: 59%

“…For example, GPCR desensitization and internalization can occur despite greatly reduced or undetectable agonist-induced phosphorylation of the receptor (Malecz et al, 1998;Sadeghi et al, 1998;Oli-vares-Reyes et al, 2001), and dephosphorylation/resensitization can occur without internalization (Gardner et al, 2001).…”

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confidence: 99%

Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

2002

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“…Our studies show for the first time that the native V2R protein exists as at least a dimer in vivo. In cell culture, dimerization of the V2R occurs early in the secretory pathway (68), although it has also been reported that formation of oligomers could be a consequence of overproduction of the protein in overexpression systems and not a required step for receptor function (53). In addition to forming homodimers, several heterodimer combinations have been reported to be formed between the V2R, the vasopressin V1a receptor (V1aR), and the oxytocin receptor (OTR), e.g., V1aR/V2R (59,61) or V2R/OTR (12,61).…”

Section: Discussionmentioning

confidence: 97%

“…Indeed, many transmembrane receptors are known to dimerize as part of their normal function, e.g., receptor tyrosine kinases (60). Previous studies in cell culture have reported that the human V2R forms homo-and heterodimers (53,61) or oligomeric complexes by domain swapping (56), and recently in an individual cell-free expression system the V2R has been shown to exist as a dimer (27). Our studies show for the first time that the native V2R protein exists as at least a dimer in vivo.…”

Section: Discussionmentioning

confidence: 99%

Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney

Fenton

Brønd²,

Nielsen

et al. 2007

American Journal of Physiology-Renal Physiology

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Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.

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“…To what extent V2 receptor oligomers are present at the cell surface has remained controversial, however. Sadeghi et al (1998) reported that V2 oligomerization was restricted to immature receptors present in the endoplasmic reticulum and that the function of the wild-type receptor remained unaltered, when co-expressed with a mutant V2 receptor lacking G-proteincoupling activity.…”

mentioning

confidence: 99%

Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer

Devost¹,

Hh²

2003

Journal of Molecular Endocrinology

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The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc-or Flag-tagged OTR respectively.Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.

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Processing and Ligand-Induced Modifications of the V2 Vasopressin Receptor

Cited by 8 publications

References 16 publications

Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney

Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer

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Processing and Ligand-Induced Modifications of the V2 Vasopressin Receptor

Cited by 8 publications

References 16 publications

Regulation of Dopamine D1Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

Regulation of Dopamine D1Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney

Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer

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Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation

Regulation of Dopamine D₁Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation