Long-term-desensitization of prostacyclin receptors is independent of the C-terminal tail

doi:10.1016/S0006-2952(03)00184-9

Biochemical Pharmacology

Volume 65, Issue 12, 15 June 2003, Pages 1991-1995

https://doi.org/10.1016/S0006-2952(03)00184-9 Get rights and content

Abstract

Persistent stimulation of the G_s protein-coupled prostacyclin receptor (IP-R) causes its slow desensitization in a variety of cell types, a significant desensitization requiring several hours. To evaluate the role of the human IP-R C-terminus in desensitization and agonist-induced internalization, a C-terminally truncated hIP-receptor was generated. The C-terminal 68 amino acid residues were deleted by introduction of a stop codon for exchange of the original S319 codon (termed D318 mutant). Wild-type (WT) and truncated receptor were expressed in COS1 cells. Pretreatment of cells with the stable prostacyclin mimetic cicaprost (200 nM) desensitized cAMP production via WT and D318 receptors to similar extents. The cAMP response of WT and D318, respectively, was reduced by approximately 50% of maximal cAMP formation after 8 hr of continuous agonist stimulation, indicating significant long-term desensitization. Moreover, agonist-promoted sequestration of WT and D318 C-terminally tagged with green fluorescent protein was demonstrated, indicating that receptor internalization was not prevented by truncation of the C-terminus. These results demonstrated that long-term desensitization and sequestration of hIP-R did not depend on structures located in the hIP-R C-terminus.

Introduction

PGI₂, the major arachidonic acid metabolite produced in vascular cells, exerts its multiple effects mostly by increased generation of cAMP subsequent to its binding to a specific IP-receptor (IP-R), which belongs to the family of GPCRs [1], [2]. PGI₂ and several mimetics have been used for a number of clinical indications including pulmonary hypertension, peripheral arterial occlusive disease and others [3], [4]. Although short-term administration of PGI₂ induced beneficial effects, they were less pronounced after more chronic use. These findings may be explained by a time-dependently reduced IP-R responsiveness to repeated agonist challenge, referred to as receptor desensitization.

Signal termination after repeated agonist challenge is a commonly observed phenomenon among GPCRs. The archetypical model for GPCR regulation involves a rapid phase of desensitization occurring within seconds to minutes after exposure to the agonist, due to agonist-induced receptor phosphorylation mediated by second messenger kinases or receptor kinases (GRKs), uncoupling the receptor from its G protein [5]. This event, referred to as short-term desensitization, is followed by sequestration of the receptor away from the cell surface [6], [7]. In contrast to most GPCR, IP-R reveals a much slower time course, significant desensitization requiring 3–10 hr [8], [9], [10]. However, the mechanisms involved in this long-term attenuation of GPCR signal transduction are still incompletely understood. Recently, a number of studies focussing on GPCRs have examined the role of the C-terminus in agonist-induced internalization and desensitization, with varying results. C-terminal truncation of β₂-ARs impaired its rapid desensitization, but a slow agonist-induced loss of β₂-receptor activity was shown to be independent of the receptor C-terminal tail [11]. The present study was designed to analyze the relevance of the IP-R C-terminus in receptor long-term desensitization. Herein, we present evidence that the C-terminal tail of hIP-R is dispensable for receptor long-term desensitization and sequestration.

Section snippets

Plasmids and site-directed mutagenesis

Complete hIP-R cDNA was kindly provided by Dr. M. Abramovitz (Merck Frosst Canada Inc.). To permit selection in mammalian cells, a 1.4 kb EcoRI fragment, containing the entire human IP-R coding sequence, was cloned into the EcoRI site of pCDNA3.1Amp (Invitrogen). The C-terminally truncated mutant was generated by introduction of a stop codon in the region coding for the intracellular tail domain using a QuikChange kit (Stratagene). The primers used were $5′- GCCCACGGAGAC TGA CAGACACCCCTTTCC -3′$

Long-term desensitization of hIP-R in COS1 cells

COS1 cells, stably expressing human IP-R (hIP-RCOS1), were assayed for cAMP generation upon treatment with the selective IP-R agonist cicaprost. Mock-transfected cells were used as a control. Basal cAMP values were similar for both cell lines with 4.7±1.5 pmol in mock-transfected cells and 5.8±1.2 pmol cAMP/mg protein in hIP-RCOS1 cells, respectively. Direct stimulation of AC with 100 nM forskolin increased cAMP formation in mock-transfected cells (15.9±2.2 pmol cAMP/mg protein) comparably to

Acknowledgements

The authors thank B. Reupert for excellent technical assistance and E. Lohmann for expert secretarial help. The work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 612, A6, B7).

References (14)

Y. Boie et al.
Cloning and expression of a cDNA for the human prostanoid IP receptor
J. Biol. Chem.
(1994)
J. Zhang et al.
Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization
J. Biol. Chem.
(1996)
J. Zhang et al.
Cellular trafficking of G protein-coupled receptor/β-arrestin endocytic complexes
J. Biol. Chem.
(1999)
A. Krane et al.
The role of protein kinase A and protein kinase C in prostanoid IP receptor desensitization in NG108-15 cells
Biochim. Biophys. Acta
(1994)
T.P. Zucker et al.
Tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E₁ in coronary artery smooth muscle cells
Eur. J. Pharmacol.
(1998)
S.M. Nilius et al.
Agonist-induced long-term desensitization of the human prostacyclin receptor
FEBS Lett.
(2000)
E.M. Smyth et al.
Phosphorylation of the prostacyclin receptor during homologous desensitization
J. Biol. Chem.
(1998)

There are more references available in the full text version of this article.

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¹: Present address: Institut für Biochemie I, Medizinische Fakultät der Universität zu Köln, Joseph-Stelzmann-Str. 52, D-50931 Köln, Germany.

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Short communicationLong-term-desensitization of prostacyclin receptors is independent of the C-terminal tail

Abstract

Introduction

Section snippets

Plasmids and site-directed mutagenesis

Long-term desensitization of hIP-R in COS1 cells

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Eur. J. Pharmacol.

FEBS Lett.

J. Biol. Chem.

Short communication
Long-term-desensitization of prostacyclin receptors is independent of the C-terminal tail