Quick M W, Simon M I, Davidson N, Lester H A, Aragay A M
Division of Biology, California Institute of Technology, Pasadena 91125.
J Biol Chem. 1994 Dec 2;269(48):30164-72.
Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.
非洲爪蟾卵母细胞被用于检测5-羟色胺1c（5HT1c）受体和促甲状腺激素释放激素（TRH）受体与内源性及异源表达的G蛋白α亚基的偶联情况。两种G蛋白偶联受体的表达均导致激动剂诱导的、Ca(2+)激活的Cl-电流，该电流通过双电极电压钳进行测量。用百日咳毒素（PTX）孵育注射后的卵母细胞，5HT诱导的Cl-电流降低了80%，而注射针对PTX敏感的Goα亚基的反义寡核苷酸可抑制50 - 65%的电流。PTX处理使TRH诱导的Cl-电流仅降低20%，但注射针对PTX不敏感的Gqα亚基的反义寡核苷酸可抑制60%的电流。注射针对一种新的非洲爪蟾磷脂酶C-β的反义寡核苷酸可抑制5HT1c（及Go）诱导的Cl-电流，而对TRH（及Gq）诱导的电流影响很小。这些结果表明，受体激活的Go和Gq与不同的效应器相互作用，很可能是磷脂酶C-β的不同同工型。还检测了每种受体与七种不同的哺乳动物G蛋白α亚基cRNAs（Goα、Goβ、Gq、G11、Gs、Golf和Gt）的共表达情况。这两种受体与前四种Gα亚基中的任何一种共表达都会使Cl-电流最大增加4 - 6倍；这种增加取决于注射的Gα亚基cRNA的量。对于与Goα和Goβ共表达的情况，这种增加被PTX阻断，而与Gq或G11共表达时则未被阻断。两种受体与Gs、Golf或Gt共表达对Ca(2+)激活的Cl-电流均无影响；此外，与Gs或Golf共表达也未能揭示5HT或TRH诱导的腺苷酸环化酶变化，这是通过囊性纤维化跨膜电导调节因子Cl-通道的激活来评估的。这些结果表明，在卵母细胞中，5HT1c和TRH受体具有以下作用：1）优先与PTX敏感的（Go）和PTX不敏感的（Gq）G蛋白偶联，且这些G蛋白作用于不同的效应器；2）在同一细胞类型内与几种不同的异源表达G蛋白α亚基偶联以激活卵母细胞的内源性Cl-电流；3）不与激活cAMP或磷酸二酯酶的G蛋白α亚基偶联。
