Voisin T, Lorinet A M, Laburthe M
Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine X. Bichat, Paris, France.
Biochem Biophys Res Commun. 1996 Aug 5;225(1):16-21. doi: 10.1006/bbrc.1996.1124.
Peptide YY (PYY)-preferring receptors are expressed in the renal proximal tubule cell clone Cl.10 isolated from the PKSV-PCT cell line. They mediate PYY-inhibited cAMP production through coupling with pertussis-sensitive Gi proteins. Previous G alpha i RNA antisense experiments demonstrated the exclusive coupling of the PYY receptor to the Gi2 protein. Here we characterized a clone stably expressing G alpha i2 antisense RNA which exhibited only a partial decrease in G alpha i2 content (#60%) as estimated by Western blot. When compared to control Cl.10 cells, this clone, referred to as Cl.10(t), exhibited: (i) an increase in the dissociation constant of PYY receptors (6.42 vs 0.63 nM); (ii) a complete absence of inhibition of [125I]PYY binding by GTP gamma S and GTP; (iii) the failure of PYY to inhibit basal and forskolin-stimulated cAMP levels; (iv) the failure of PYY to stimulate [35S]GTP gamma S binding to membranes. These findings show that partial knockdown of G alpha i2 expression in Cl.10 cells completely abolish the coupling of PYY receptors to biological response.
从PKSV-PCT细胞系分离出的肾近端小管细胞克隆Cl.10中表达了对肽YY（PYY）有偏好的受体。它们通过与对百日咳敏感的Gi蛋白偶联来介导PYY抑制的cAMP产生。先前的Gαi RNA反义实验证明了PYY受体与Gi2蛋白的特异性偶联。在此，我们鉴定了一个稳定表达Gαi2反义RNA的克隆，通过蛋白质印迹法估计，该克隆的Gαi2含量仅部分降低（60%）。与对照Cl.10细胞相比，这个称为Cl.10(t)的克隆表现出：（i）PYY受体解离常数增加（6.42对0.63 nM）；（ii）GTPγS和GTP完全不抑制[125I]PYY结合；（iii）PYY不能抑制基础和福斯可林刺激的cAMP水平；（iv）PYY不能刺激[35S]GTPγS与膜结合。这些发现表明，Cl.10细胞中Gαi2表达的部分敲低完全消除了PYY受体与生物学反应的偶联作用。
