Wietfeld Doreen, Heinrich Nadja, Furkert Jens, Fechner Klaus, Beyermann Michael, Bienert Michael, Berger Hartmut
Institute of Molecular Pharmacology, Robert-Rössle-Strasse 10, Berlin 13125, Germany.
J Biol Chem. 2004 Sep 10;279(37):38386-94. doi: 10.1074/jbc.M405335200. Epub 2004 Jul 12.
The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC(50) values as measured by stimulation of [(35)S]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [(35)S]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11)in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i) -mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.
研究了人胚肾（HEK）293（HEK-rCRFR1）细胞膜中大鼠促肾上腺皮质激素释放因子受体1型（rCRFR1）对G蛋白激活的调节作用。对应于高亲和力和低亲和力配体结合位点，蛙皮素和其他肽类CRFR1配体分别引起G蛋白激活的高效能和低效能反应，通过刺激[(35)S]GTPγS结合测得的EC(50)值相差64倍。与低效能反应相反，高效能反应的GTPγS亲和力较低，对百日咳毒素（PTX）不敏感，且发生同源脱敏。当腺苷酸环化酶的高效能刺激被消除且其活性被蛙皮素低效能抑制时，在腺苷酸环化酶活性中也观察到了明显的脱敏现象。根据这些结果以及对[(35)S]GTPγS结合的Gα(s)和Gα(i)亚基的免疫沉淀，得出结论：高效能和低效能[(35)S]GTPγS结合刺激分别反映了与G(s)和G(i)蛋白的偶联，只有G(s)偶联发生同源脱敏。对[(35)S]GTPγS结合的Gα(q/11)的免疫沉淀揭示了与Gα(q/11)的额外偶联，其也发生同源脱敏。尽管Gα(q/11)偶联对PTX不敏感，但细胞中蛙皮素刺激的肌醇磷酸积累的一半对PTX敏感，这表明除了Gα(q/11)外，Gα(i)也参与了肌醇代谢的刺激。得出结论：CRFR1通过至少两种不同方式进行信号传导，一种导致G(s)和Gα(q/11)介导的信号传导步骤和脱敏，另一种导致Gα(i)介导的信号而不发生脱敏。此外，刺激配体和GTP的浓度以及脱敏可能是决定CRFR1与不同G蛋白偶联实际比例的调节机制的一部分。
