Elsevier

Cellular Signalling

Volume 11, Issue 10, October 1999, Pages 743-751
Cellular Signalling

Homologous and Heterologous Phosphorylation of the Vasopressin V1a Receptor

https://doi.org/10.1016/S0898-6568(99)00035-2Get rights and content

Abstract

The vasopressin V1a receptor undergoes homologous and heterologous desensitizations which can be mimicked by activation of protein kinase C. This suggests that phosphorylation of the V1a receptor may be involved in the desensitization mechanisms. Such a phosphorylation was presently investigated in HEK 293 cells stably transfected with rat vasopressin V1a receptor. Metabolic labelling and immunoprecipitation of epitope-tagged V1a receptor evidenced a 52-kDa band and a 92-kDa band. Glycosidase treatments and immunoblotting experiments suggest that the 52-kDa band corresponds to an immature unprocessed receptor protein, whereas the 92-kDa band would correspond to a highly glycosylated form of the mature V1a receptor. Exposure of the cells to vasopressin induced a selective 32P phosphate incorporation in the 92-kDa form of the receptor. This homologous ligand-induced phosphorylation was dose dependent with maximal phosphate incorporation corresponding to four times the basal level. Stimulation of the endogenous phospholipase C-coupled m3 muscarinic receptor by carbachol-induced heterologous phosphorylation of the V1a receptor whose amplitude was half that of the homologous phosphorylation. This heterologous phosphorylation was associated with a reduced vasopressin-dependent increase in intracellular calcium.

Introduction

Most of the G-protein-coupled receptors undergo homologous desensitization when stimulated with their own ligand. This is characterized by refractoriness of the receptor to a second hormonal stimulation, and mainly corresponds to a decoupling between receptor and G proteins. It has been proposed that the molecular mechanisms underlying such acute desensitization involve phosphorylation of the serine/threonine residues of the intracellular domains of the receptor [1]. Phosphorylation may be promoted by activation of protein kinase A (PKA) for the Gs and adenylyl-cyclase coupled receptors, or protein kinase C (PKC) for the Gq and phospholipase-coupled receptors. Phosphorylation may also be induced by receptor kinase (GRKs) consecutive to agonist–receptor interaction [2]. These phosphorylation pathways are not exclusive and can be shared by the same receptor [3]. In addition to homologous desensitization, some G-protein-coupled receptors exhibit heterologous desensitization consecutive to stimulation of distinct receptors within the same cells. This heterologous desensitization may often be reproduced by an increase in intracellular messengers and is also thought to be dependent on phosphorylation of the receptor. However, recent experiments on the vasopressin V2 receptor indicated that, although exposure to vasopressin promotes phosphorylation and desensitization, mutated V2 receptor lacking their phosphorylation sites still exhibited homologous desensitization [4]. The authors concluded that abolishing phosphorylation reduced the extent of desensitization but did not block it.

Relationships between phosphorylation and desensitization are less documented for the vasopressin V1a receptor. This receptor, widely distributed in organs such as brain, blood vessels, kidneys, testis, and liver, is coupled to Gq protein and to the phospholipase C pathway [5]. When stimulated by vasopressin, it induces an increase in intracellular inositol phosphate, a transient calcium peak and an activation of protein kinase C. Exposure of the V1a receptor to vasopressin results in rapid homologous desensitization which has been evidenced in native tissues as in heterologous expression systems 6, 7, 8, 9. This desensitization may be reproduced by direct activation of cytosolic protein kinase C by diacylglycerol, or by intracellular injection of catalytic subunit of protein kinase C. Similarly, stimulation of endogenous phospholipase C-coupled-muscarinic receptors in cells expressing the V1a receptor results in heterologous desensitization [8]. These experiments suggests that the protein kinase C-dependent phosphorylation of the V1a receptor is implicated in receptor desensitization.

Such homologous and heterologous phosphorylations of the V1a receptor were presently investigated. TagHA epitopes were inserted in cDNA of rat V1a receptor to allow its extraction and immunoprecipitation. The epitope-tagged receptor was stably transfected in HEK 293 cells and its binding ability and calcium signalling were characterized. The native and glycosylated forms of the V1a receptor present in the transfected cells were defined by metabolic labelling, Western blot analysis, and selective digestion by glycosidases. The homologous agonist-induced phosphorylation of the receptor was quantified by incorporation of 32P phosphate. Heterologous phosphorylation was investigated by stimulation of the endogenous phospholipase C-linked muscarinic receptor.

Section snippets

Chemicals

DMEM, methionine-free DMEM, phosphate-free DMEM, geneticin G418, and fetal bovine serum, were from Life Technologies, Inc.; 35S-Express protein labelling mix, [32P]H3PO4 in water (pH 5–7) and [3H]Arginine vasopressin were from Dupont NEN. Fura 2 was from Molecular Probes, penicillin-streptomycin from Boehringer Mannheim, arginine vasopressin from Neosystem, and okadaic acid from Calbiochem (France Biochem). Superfect reagents were from Quiagen. Cell culture materials were from Dutcher. Bovine

Results and discussion

Two stably transfected HEK 293 cell lines containing rat wild type or epitope-tagged V1a receptors cDNA were established and characterized by binding experiments. The number of 3H vasopressin binding sites per cell measured in saturation conditions was close to 150,000 in the HEK 293 cells transfected with the V1a wild type receptor (HEK 293 V1aWT) or with the epitope-tagged V1a receptor (HEK 293 V1aTagHA). Kd values calculated from Scatchard plots were 0.59 ± 0.13 nM (n = 4) and 0.50 ± 0.07 nM

Acknowledgements

We are grateful to Marion Lambert for her technical assistance in calcium determinations.

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