Wang S, Hashemi T, Fried S, Clemmons A L, Hawes B E
Department of CNS/CV Biological Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Biochemistry. 1998 May 12;37(19):6711-7. doi: 10.1021/bi9728405.
The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.
神经肽甘丙肽发挥的多种生理功能可能受多种G蛋白偶联受体亚型和细胞内信号通路的调控。最近已克隆出三种甘丙肽受体亚型（GalRs），但这些受体的G蛋白偶联情况尚未完全明确。我们构建了表达GalR1和GalR2的中国仓鼠卵巢（CHO）细胞系，并系统研究了这两种受体与Gs、Gi、Go和Gq蛋白偶联的可能性。甘丙肽未刺激GalR1/CHO或GalR2/CHO细胞中cAMP水平升高，提示这两种受体均无法与Gs偶联。甘丙肽使GalR1/CHO细胞中福斯高林刺激的cAMP生成量降低70%，使GalR2/CHO细胞中降低30%，提示GalR1与Gi强烈偶联，GalR2与Gi的偶联较弱。GalR1和GalR2均介导百日咳毒素敏感的丝裂原活化蛋白激酶（MAPK）活性（升高2 - 3倍）。GalR1介导的刺激可被β - 肾上腺素能受体激酶C末端（βARKct）的表达抑制，βARKct可特异性抑制Gβγ信号传导，但不受蛋白激酶C（PKC）抑制剂双[吲哚基马来酰亚胺]或细胞内PKC耗竭的影响。相比之下，GalR2介导的MAPK活化不受βARKct表达的影响，但可被PKC活性抑制所消除。数据表明，GalR1与Gβγ信号通路偶联以介导MAPK活化。相比之下，GalR2利用不同的信号通路介导MAPK活化，这与CHO细胞中Go介导的MAPK活化一致。甘丙肽无法刺激表达GalR1的CHO或COS - 7细胞中肌醇磷酸（IP）积累。相比之下，甘丙肽使表达GalR2的CHO或COS - 7细胞中IP生成量增加7倍。GalR2介导的IP生成不受百日咳毒素影响，提示GalR2与Gq/G11相关联。因此，GalR1受体似乎仅激活Gi途径。相比之下，GalR2能够刺激与Go、Gq/G11和Gi活化一致的信号传导。GalR1和GalR2不同的信号传导情况及组织分布模式可能是这些甘丙肽受体介导的甘丙肽作用功能谱的基础，并调节甘丙肽的多种生理功能。
