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首页> 外文期刊>Cellular Signalling >Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1 beta and response to PKC phosphorylation
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Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1 beta and response to PKC phosphorylation

机译：结构域决定CRH受体1型变异R1 beta的信号传导特征以及对PKC磷酸化的反应

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摘要

Mammalian adaptive mechanisms to stressful stimuli involve release of corticotropin-releasing hormone (CRH) and downstream activation of specific G-protein-coupled 7 transmembrane domain receptors. These CRH receptors (CRH-R) are expressed as multiple mRNA spliced variants. In contrast to other mammals, the human type 1 CRH-R gene contains an additional exon (exon 6) that needs to be spliced out in order to generate the fully active CRH-R1 alpha. Transcription of all 14 exons results in a CRH-R1 variant (CRH-R1 beta) with an extended 1st intracellular loop (IC1); this sequence modification impairs signalling activity and alters receptor responsiveness to PKC-induced phosphorylation that leads to signalling desensitization and receptor endocytosis. To elucidate structure-function relationships and delineate sequences involved in CRH-R1 beta properties, site directed mutagenesis was used to introduce a number of specific mutations into IC1 of CRH-R1 beta as well as replace specific phospho-acceptor residues within the aminoacid sequence of CRH-R1 alpha and CRH-R1 beta Mutant receptors were transiently expressed in human embryonic kidney (HEK293) cells and tested for their abilities to increase intracellular cAMP and their response to PKC-induced phosphorylation. Results identified a penta-aminoacid cassette within the 29-aminoacid insert of CRH-R1 beta, which contains multiple positive charged aminoacids (F-170-R-174), as an important structural determinant for the impaired cAMP response. Furthermore, serine at position 408 in the carboxy-termimis appears to be important for mediating CRI-R1 alpha resistance, but not CRH-R1 beta susceptibility, to PKC-induced desensitization and internalization. These findings provide new insights about the structural determinants of CRH-R1 coupling to Gs proteins and response to protein kinase phosphorylation. (C) 2007 Elsevier Inc. All rights reserved.
机译：哺乳动物对应激刺激的适应性机制包括释放促肾上腺皮质激素释放激素（CRH）和下游激活特定的G蛋白偶联的7个跨膜结构域受体。这些CRH受体（CRH-R）被表达为多个mRNA剪接变体。与其他哺乳动物相反，人类1型CRH-R基因包含一个额外的外显子（6号外显子），需要剪接该外显子才能生成具有完全活性的CRH-R1α。全部14个外显子的转录会导致CRH-R1变体（CRH-R1 beta）带有扩展的第一个细胞内环（IC1）;该序列修饰损害信号传导活性并改变受体对PKC诱导的磷酸化的响应性，从而导致信号脱敏和受体内吞。为了阐明CRH-R1 beta特性所涉及的结构-功能关系和序列，使用了定点诱变技术将许多特定突变引入CRH-R1 beta IC1中，并替换了CRH-R1 beta氨基酸序列中的特定磷酸受体残基。 CRH-R1α和CRH-R1β突变受体在人类胚胎肾脏（HEK293）细胞中瞬时表达，并测试其增加细胞内cAMP的能力以及对PKC诱导的磷酸化的反应。结果鉴定出CRH-R1 beta的29个氨基酸插入物中的五氨基酸盒，其中包含多个带正电荷的氨基酸（F-170-R-174），是导致cAMP反应受损的重要结构决定因素。此外，在羧基末端的位置408处的丝氨酸对于介导CRI-R1α对PKC引起的脱敏和内在化的敏感性而不是CRH-R1β敏感性很重要。这些发现为CRH-R1与Gs蛋白偶联的结构决定簇以及对蛋白激酶磷酸化的反应提供了新的见解。 （C）2007 Elsevier Inc.保留所有权利。

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