The C‐terminal domain of the Gs‐coupled EP4 receptor confers agonist‐dependent coupling control to Gi but no coupling to Gs in a receptor hybrid with the Gi‐coupled EP3 receptor

FEBS Letters

Hänecke, Kristina¹  Jungermann, Kurt¹  Neuschäfer-Rube, Frank¹  Blaschke, Volker¹  Püschel, Gerhard P¹ 

[1] Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, Georg-August-Universität, 37073 Göttingen, Germany

关键词: Prostaglandin receptor;    Chimeric receptor;    G-protein coupling;    CHO cells;    Chinese hamster ovary cells;    DMEM;    Dulbecco's modified Eagle medium;    EPR;    E-prostaglandin receptor;    FCS;    fetal calf serum;    HAM-F12;    nutrient mixture Ham's F-12;    HEK cells;    human embryonal kidney cells;    IBMX;    3-isobutyl-1-methylxanthine;    MBS;    modified bovine serum;    MEM;    minimal essential medium;    MES;    4-morpholine-ethanesulfonic acid;    PCR;    polymerase chain reaction;    PG;    prostaglandin;    PTX;    pertussis toxin;   

DOI  :  10.1016/S0014-5793(96)01468-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Prostaglandin E₂ receptors (EPR) belong to the family of G-protein-coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G-proteins. EP₁R are coupled to G_q, EP₂ and EP₄R to G_s and EP₃R to G_i. Different C-terminal splice variants of the bovine EP₃R are coupled to different G-proteins. A mouse EP₃R whose C-terminal domain had been partially truncated no longer showed agonist-induced G_i-protein activation and was constitutively active. In order to test the hypothesis that the C-terminal domain confers coupling specificity of the receptors on the respective G-proteins, a cDNA for a hybrid rEP₃hEP₄R, containing the N-terminal main portion of the G_i-coupled rat EP_3βR including the 7th transmembrane domain and the intracellular C-terminal domain of the G_s-coupled human EP₄R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP₃hEP₄R cDNA expressed a plasma membrane PGE₂ binding site with a slightly lower K _d value for PGE₂ but an identical binding profile for receptor-specific ligands as cells transfected with the native rat EP_3βR. In HepG₂ cells stably transfected with the chimeric rEP₃hEP₄R cDNA PGE₂ did not increase cAMP formation characteristic of G_s coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of G_i coupling. This effect was inhibited by pre-treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP_3βR. Apparently, the intracellular C-terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.

【 授权许可】

Unknown