Voisin T, Lorinet A M, Maoret J J, Couvineau A, Laburthe M
Unité de Recherche de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier Bichat, Paris, France.
J Biol Chem. 1996 Jan 5;271(1):574-80. doi: 10.1074/jbc.271.1.574.
A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from the PKSV-PCT renal proximal tubule cell line which expressed peptide YY (PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A., and Laburthe, M. (1993) J. Biol. Chem. 268, 20547-20554). In order to identify G(i) protein(s) coupled to PYY receptors, antisense G alpha i protein RNAs were expressed in Cl.10 cells by transfecting the pcDNA3 vector into which were inserted 39 bases of the 5'-noncoding region of G alpha i2 or G alpha i3 used as specific antisense templates. A Cl.10/alpha i2-clone was selected which displayed a drastic decrease (> 90%) of the expression of G alpha i2 without changes of G alpha i3, G alpha s, and G beta subunits (G alpha i1 is not present in Cl.10 cells) as evidenced by Western blots. When compared to untransfected cells, this clone exhibited: (i) an increase in the dissociation constant of PYY receptors (5.3 versus 0.6 nM) identical to that observed in pertussis toxin-treated untransfected cells; (ii) an absence of inhibition of 125I-PYY binding by guanosine 5'-O-(thiotriphosphate) (GTP gamma S); and (iii) the failure of PYY to inhibit cAMP levels and to stimulate [methyl-3H]thymidine incorporation into DNA. A clone was also selected which exhibited a specific decrease (> 80%) of G alpha i3 as compared to untransfected cells. The sensitivity to GTP gamma S and the dissociation constant of PYY receptors as well as PYY-mediated inhibition of cAMP were identical to those observed in untransfected cells. These findings support an exclusive coupling of PYY receptors to G alpha i2.
从表达肽YY（PYY）受体的PKSV - PCT肾近端小管细胞系中筛选出一个名为Cl.10的克隆PKSV - PCT Cl.10（瓦赞，T.，本斯，M.，克吕佐，F.，万德瓦勒，A.，和拉比特，M.（1993年）《生物化学杂志》268卷，20547 - 20554页）。为了鉴定与PYY受体偶联的G(i)蛋白，通过转染插入了用作特异性反义模板的Gαi2或Gαi3的5'-非编码区39个碱基的pcDNA3载体，在Cl.10细胞中表达反义Gαi蛋白RNA。筛选出一个Cl.10/αi2克隆，经蛋白质免疫印迹法证实，该克隆显示Gαi2的表达急剧下降（>90%），而Gαi3、Gαs和Gβ亚基无变化（Cl.10细胞中不存在Gαi1）。与未转染的细胞相比，该克隆表现出：（i）PYY受体解离常数增加（5.3对0.6 nM），与在百日咳毒素处理的未转染细胞中观察到的相同；（ii）5'-O-(硫代三磷酸)鸟苷（GTPγS）对125I - PYY结合无抑制作用；（iii）PYY未能抑制环磷酸腺苷（cAMP）水平以及刺激[甲基-3H]胸苷掺入DNA。还筛选出一个与未转染细胞相比Gαi3特异性下降（>80%）的克隆。对GTPγS的敏感性、PYY受体的解离常数以及PYY介导的cAMP抑制作用与在未转染细胞中观察到的相同。这些发现支持PYY受体与Gαi2的排他性偶联。
